Probes for Following Receptor Binding and Phagocytosis—Section 16.1

Page Contents

• Ligands for Studying Receptor-Mediated Endocytosis
• Methods for Detecting Internalized Fluorescent Ligands
• Membrane Markers of Endocytosis and Exocytosis
• High Molecular Weight Polar Markers
• Low Molecular Weight Polar Markers
• Data Table
• Ordering Information

The plasma membrane defines the inside and outside of the cell. It not only encloses the cytosol to maintain the intracellular environment but also serves as a formidable barrier to the extracellular environment. Because cells require input from their surroundings—in the form of hydrated ions, small polar molecules, large biomolecules and even other cells—they have developed strategies for overcoming this barrier. Some of these mechanisms involve initial formation of receptor–ligand complexes, followed by transport of the ligand across the cell membrane.

This section focuses on probes for following receptor binding, endocytosis and exocytosis. Probes for Neurotransmitter Receptors—Section 16.2 describes tools for studying neurotransmitter receptors, which mediate external chemical messenger control over the electrical activity of neurons. Probes for Ion Channels and Carriers—Section 16.3 discusses strategies for monitoring ion channels and carriers, which are the molecular centerpiece of neural transmission and bioenergetics.

We offer a variety of fluorescent and fluorogenic ligands that bind to membrane receptors and are subsequently internalized. In some cases, the bound ligand is released intracellularly and the receptor is then recycled to the plasma membrane. Receptor binding may also result in signal transduction (Probes for Signal Transduction—Chapter 17), Ca2+ mobilization (Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19), intracellular pH changes (pH Indicators—Chapter 20) and formation of reactive oxygen species (ROS, Probes for Reactive Oxygen Species, Including Nitric Oxide—Chapter 18).

Fc OxyBURST Green Assay Reagent: Fluorogenic Immune Complex

When soluble or surface-bound IgG immune complexes interact with Fc receptors on phagocytic cells, a number of host defense mechanisms are activated, including phagocytosis and activation of an NADPH oxidase–mediated oxidative burst. Dichlorodihydrofluorescein diacetate (H2DCFDA, D399; Generating and Detecting Reactive Oxygen Species—Section 18.2), a cell-permeant fluorogenic probe that localizes in the cytosol, has frequently been used to monitor this oxidative burst. Its fluorescence response, however, is limited by the diffusion rate of the reactive oxygen species into the cytosol from the phagovacuole where it is generated. In contrast, Fc OxyBURST assay reagents permit direct measurement of the kinetics of Fc receptor–mediated internalization and the subsequent oxidative burst in the phagovacuole, yielding signals that are many times brighter than those generated by H2DCFDA (Figure 16.1.1, Figure 16.1.2).

Fc OxyBURST Green assay reagent (F2902) was developed in collaboration with Elizabeth Simons of Boston University to monitor the oxidative burst in phagocytic cells using fluorescence instrumentation. Fc OxyBURST Green assay reagent comprises bovine serum albumin (BSA) that has been covalently linked to dichlorodihydrofluorescein (H2DCF) and then complexed with a purified rabbit polyclonal anti-BSA antibody (A11133). When these immune complexes bind to Fc receptors, the nonfluorescent H2DCF molecules are internalized within the phagovacuole and subsequently oxidized to green-fluorescent 2',7'-dichlorofluorescein (DCF; Figure 16.1.1, Figure 16.1.2). Unlike H2DCFDA, Fc OxyBURST Green assay reagent does not require intracellular esterases for activation, making this reagent particularly suitable for detecting the oxidative burst in cells with low esterase activity such as monocytes. Fc OxyBURST Green assay reagent reportedly produces >8 times more fluorescence than does H2DCFDA at 60 seconds and >20 times more at 15 minutes following internalization of the immune complex.

Published reports have described the use of Fc OxyBURST Green assay reagent to study the oxidative burst in phagovacuoles. Neutrophils from patients with chronic granulomatous disease, a genetic deficiency known to disable NADPH oxidase–mediated oxidative bursts, were observed to bind but not oxidize Fc OxyBURST Green assay reagent  (Figure 16.1.3). Using microfluorometry to detect the Fc OxyBURST Green signal, researchers were able to simultaneously monitor oxidative activity and membrane currents in voltage-clamped human mononuclear cells.

OxyBURST Green H₂HFF BSA Reagent

OxyBURST Green H₂HFF BSA reagent (O13291) is similar to Fc OxyBURST Green assay reagent, except that it is prepared by reacting the succinimidyl ester of a reduced form of our Oregon Green 488 dye with BSA. The absorption maximum of the oxidation product of this reagent (~492 nm) matches the 488 nm spectral line of the argon-ion laser better than does that of Fc OxyBURST Green assay reagent (~495 nm). OxyBURST Green H₂HFF BSA reagent can also be complexed with anti-BSA antibody to form an immune complex that can be utilized like the Fc OxyBURST Green assay reagent (F2902, see above).

All of the OxyBURST reagents are slowly oxidized by molecular oxygen and are also susceptible to oxidation catalyzed by illumination in a fluorescence microscope. These reagents are reasonably stable in solution for at least six months when stored under nitrogen or argon in the dark at 4°C. We also offer a purified rabbit polyclonal anti-BSA antibody (A11133), which can bind any of our BSA conjugates (Protein Conjugates—Section 14.7) or fluorogenic DQ BSA conjugates (D12050, D12051; Detecting Peptidases and Proteases—Section 10.4) to create immune complexes for analyzing the Fc receptor–mediated phagocytosis pathway. In the case of the anti-BSA antibody complex with DQ BSA, initial binding and internalization of the probe is followed by hydrolysis to fluorescent peptides by proteases in the phagovacuole (Figure 16.1.4).

Amine-Reactive OxyBURST Green Reagent

As an alternative to Fc OxyBURST Green assay reagent and OxyBURST Green H2HFF BSA, we offer amine-reactive OxyBURST Green H2DCFDA succinimidyl ester (2',7'-dichlorodihydrofluorescein diacetate, SE; D2935), which can be used to prepare oxidation-sensitive conjugates of a wide variety of biomolecules and particles, including antibodies, antigens, peptides, proteins, dextrans, bacteria, yeast and polystyrene microspheres. Following conjugation to amines, the two acetates of OxyBURST Green H2DCFDA reagent can be removed by treatment with hydroxylamine at near-neutral pH to yield the oxidant-sensitive dichlorodihydrofluorescein conjugates. Thus, like our Fc OxyBURST Green assay reagent, they provide a means of detecting the oxidative burst in phagocytic cells.

Several other reagents—dihydrofluoresceins, dihydrorhodamines, dihydroethidium and chemiluminescent probes—that have been used to detect the reactive oxygen species (ROS) produced during phagocytosis are described in Generating and Detecting Reactive Oxygen Species—Section 18.2.

Fluorescent Low-Density Lipoprotein Complexes

The human LDL complex, which delivers cholesterol to cells by receptor-mediated endocytosis, comprises a core of about 1500 molecules of cholesteryl ester and triglyceride, surrounded by a 20 Å–thick shell of phospholipids, unesterified cholesterol and a single copy of apoprotein B 100  (MW ~500,000 daltons). Once internalized, LDL dissociates from its receptor and eventually appears in lysosomes. In addition to unlabeled LDL (L3486), which has been reported to be an effective vehicle for selectively delivering antitumor drugs to cancer cells, we offer two classes of labeled LDL probes—those containing an unmodified apoprotein, used to study the mechanisms of normal cholesterol delivery and internalization, and those with an acetylated apoprotein, used to study endothelial, microglial and other cell types that express receptors that specifically bind this modified LDL (see below).

For the class of labeled LDL probes containing unmodified apoprotein, we prepare LDL noncovalently labeled with either DiI (DiI LDL, L3482) or the BODIPY FL fluorophore (BODIPY FL LDL, L3483), highly fluorescent lipophilic dyes that diffuse into the hydrophobic portion of the LDL complex without affecting the LDL-specific binding of the apoprotein. As compared with DiI LDL, BODIPY FL LDL is more efficiently excited by the 488 nm spectral line of the argon-ion laser, making it better suited for flow cytometry and confocal laser-scanning microscopy studies. Like our BODIPY FL C5-ceramide (D3521, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4), BODIPY FL LDL may exhibit concentration-dependent long-wavelength emission (>550 nm), precluding its use for multicolor labeling with red fluorophores. Both DiI LDL and BODIPY FL LDL have been used to investigate the binding specificity and partitioning of LDL throughout the Schistosoma mansoni parasite  (). Fluorescent LDL complexes have also proven useful in a variety of experimental systems to:

• Count the number of cell-surface LDL receptors, analyze their motion and clustering and follow their internalization 
• Demonstrate that fibroblasts grown continuously in the presence of DiI LDL (L3482) proliferate normally and exhibit normal morphology, making DiI LDL a valuable alternative to 125I-labeled LDL for quantitating LDL receptor activity 
• Identify LDL receptor–deficient Chinese hamster ovary (CHO) cell mutants 
• Image LDL receptor endocytosis in COS7 cells expressing green-fluorescent protein (GFP)–tagged GTPase 
• Investigate the modulation of LDL receptor expression by statin drugs

We prepare fluorescent LDL products from fresh human plasma, and they should be stored refrigerated and protected from light; LDL products must not be frozen. Because preparation of these complexes involves several variables, some batch-to-batch variability in degree of labeling and fluorescence yield is expected.

Fluorescent Acetylated LDL Complexes

If the lysine residues of LDL's apoprotein have been acetylated, the LDL complex no longer binds to the LDL receptor, but rather is taken up by macrophage and endothelial cells that possess "scavenger" receptors specific for the modified LDL. Once the acetylated LDL (AcLDL) complexes accumulate within these cells, they assume an appearance similar to that of foam cells found in atherosclerotic plaques. We offer unlabeled AcLDL (L35354), as well as AcLDL noncovalently labeled with DiI (L3484) and AcLDL covalently labeled with Alexa Fluor 488 dye (L23380), Alexa Fluor 594 dye (L35353) or BODIPY FL dye (L3485). Fluorescent dye conjugates of high-density lipoproteins, including one prepared using Alexa Fluor 488 succinimidyl ester (A20000, A20100; Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), are taken up via the same receptor as acetylated LDL complexes.

Using DiI AcLDL, researchers have discovered that the scavenger receptors on rabbit fibroblasts and smooth muscle cells appear to be up-regulated through activation of the protein kinase C pathway. DiI AcLDL has also been used to show that Chinese hamster ovary (CHO) cells express AcLDL receptors that are distinct from macrophage scavenger receptors. Ultrastructural localization of endocytic compartments that maintain a connection to the extracellular space has been achieved by photoconversion of DiI AcLDL in the presence of diaminobenzidine  (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). A quantitative assay for LDL- and scavenger-receptor activity in adherent and nonadherent cultured cells that avoids the use of both radioactivity and organic solvents has been described.

It has now become routine to identify endothelial cells and microglial cells in primary cell culture by their ability to take up DiI AcLDL  (). DiI AcLDL was employed in order to confirm endothelial cell identity in investigations of shear stress  and P-glycoprotein expression, as well as to identify blood vessels in a growing murine melanoma. In addition, patch-clamp techniques have been used to investigate membrane currents in mouse microglia, which were identified both in culture and in brain slices by their staining with DiI AcLDL. For some applications, Alexa Fluor 488, Alexa Fluor 594 and BODIPY FL AcLDL may be the preferred probes because the dyes are covalently bound to the modified apoprotein portion of the LDL complex and are therefore not extracted during subsequent manipulations of the cells. Furthermore, the green-fluorescent Alexa Fluor 488 AcLDL has spectral characteristics similar to fluorescein and is useful for analyses with instruments equipped with the 488 nm argon-ion laser excitation sources, including flow cytometers and confocal laser-scanning microscopes. The bright and photostable red-fluorescent Alexa Fluor 594 AcLDL conjugate is useful for multilabeling experiments with green-fluorescent probes and can be efficiently excited by the 594 nm spectral line of the orange He-Ne laser.

Fluorescent Lipopolysaccharides

We offer fluorescent conjugates of lipopolysaccharides (LPS) from Escherichia coli and Salmonella minnesota (Fluorescent lipopolysaccharide conjugates—Table 16.1), including:

• Alexa Fluor 488 LPS from E. coli serotype 055:B5 (L23351)
• Alexa Fluor 488 LPS from S. minnesota (L23356)
• Alexa Fluor 568 LPS from E. coli serotype 055:B5 (L23352)
• Alexa Fluor 594 LPS from E. coli serotype 055:B5 (L23353)
• BODIPY FL LPS from E. coli serotype 055:B5 (L23350)

LPS, also known as endotoxins, are a family of complex glycolipid molecules located on the surface of gram-negative bacteria. LPS play a large role in protecting the bacterium from host defense mechanisms and antibiotics. Binding of LPS to the CD14 cell-surface receptor of phagocytes is the key initiation step in the mammalian immune response to infection by gram-negative bacteria. The structural core of LPS, and the primary determinant of its biological activity, is an N-acetylglucosamine derivative, lipid A (Figure 16.1.5). In many gram-negative bacterial infections, LPS are responsible for clinically significant symptoms like fever, low blood pressure and tissue edema, which can lead to disseminated intravascular coagulation, organ failure and death.

The fluorescent BODIPY FL and Alexa Fluor LPS conjugates, which are labeled with succinimidyl esters of these dyes, allow researchers to follow LPS-elicited inflammatory responses. Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes. Alexa Fluor 488 LPS conjugates (L23351, L23356) have been shown to selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes and microglia.

The BODIPY FL derivative of LPS from E. coli strain LCD25 (L23350) was used to measure the transfer rate of LPS from monocytes to high-density lipoprotein  (HDL). Another study utilized a BODIPY FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi apparatus in neutrophils, although this could have been due to probe metabolism. It has been reported that organelles other than the Golgi are labeled by some fluorescent or nonfluorescent LPS. Cationic lipids are reported to assist the translocation of fluorescent lipopolysaccharides into live cells; cell surface–bound LPS can be quenched by trypan blue.

Other probes useful for analyzing lipopolysaccharides include fluorescent analogs of the LPS-binding antibiotic polymyxin B (Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3) and BODIPY TR cadaverine (D6251, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4). BODIPY TR cadaverine binds with high selectivity to lipid A, forming the basis for high-throughput ligand displacement assays for identifying endotoxin antagonists.

Epidermal Growth Factor Derivatives

Epidermal growth factor (EGF) is a 53–amino acid polypeptide hormone (MW 6045 daltons) that stimulates division of epidermal and other cells. The EGF receptors include the HER-2/neu receptor (where "HER-2" is an acronym for human epidermal growth factor receptor-2 and "neu" refers to an original mouse origin); HER-2/neu overexpression has evolved as a prognostic/predictive factor in some solid tumors. Binding of EGF to its 170,000-dalton receptor protein results in the activation of kinases, phospholipases and Ca²⁺ mobilization and precipitates a wide variety of cellular responses related to differentiation, mitogenesis, organ development and cell motility.

We offer unlabeled mouse submaxillary gland EGF (E3476), as well as the following EGF conjugates, each containing a single fluorophore or biotin on the N-terminal amino acid:

• Fluorescein EGF (E3478)
• Oregon Green 514 EGF (E7498)
• Tetramethylrhodamine EGF (E3481)
• Biotin-XX EGF (E3477)

The dissociation constant of the EGF conjugates in DMEM-HEPES medium is in the low nanomolar range for human epidermoid carcinoma (A431) cells, a value that approximates that of the unlabeled EGF. Fluorescently labeled EGF has enabled scientists to use fluorescence resonance energy transfer techniques to assess EGF receptor–receptor and receptor–membrane interactions  (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Using fluorescein EGF as the donor and tetramethylrhodamine EGF as the acceptor, researchers examined temperature-dependent lateral and transverse distribution of EGF receptors in A431 cell plasma membranes. When fluorescein EGF binds to A431 cells, it apparently undergoes a biphasic quenching, which can be attributed first to changes in rotational mobility upon binding and then to receptor–ligand internalization. By monitoring this quenching in real time, the rate constants for the interaction of fluorescein EGF with its receptor were determined. Although fluorescently labeled EGF can be used to follow lateral mobility and endocytosis of the EGF receptor, the visualization of fluorescent EGF may require low-light imaging technology or Qdot nanocrystals, especially in cells that express low levels of the EGF receptor. In cells with few EGF receptors, it can be difficult to detect signal over background fluorescence unless signal amplification methods are employed (Figure 16.1.6).

Biotin-XX EGF contains a long spacer arm that enhances the probe's affinity for the EGF receptor and facilitates binding of dye-, Qdot nanocrystal– or enzyme-conjugated streptavidins  (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). Using biotinylated EGF and phycoerythrin-labeled secondary reagents (Phycobiliproteins—Section 6.4), researchers were able to detect as few as 10,000 EGF cell-surface receptors by confocal laser-scanning microscopy. Tyramide signal amplification (TSA) technology (Peroxidase-Based Signal Amplification, Including TSA—Section 6.2) is particularly valuable for detection and localization of low-abundance EGF receptors by both imaging and flow cytometry (Figure 16.1.6). For additional sensitivity, we prepare biotinylated EGF precomplexed to fluorescent streptavidin:

• Biotinylated EGF complexed to Alexa Fluor 488 streptavidin (E13345, )
• Biotinylated EGF complexed to Alexa Fluor 555 streptavidin (E35350)
• Biotinylated EGF complexed to Alexa Fluor 647 streptavidin (E35351)
• Biotinylated EGF complexed to Texas Red streptavidin  (E3480).

These products yield severalfold brighter signals per EGF receptor when compared with the direct conjugates. We have found that EGF receptors can easily be detected with these complexes without resorting to low-light imaging technology (). A quantitative high-content screening (HCS) assay for EGF receptor modulators based on imaging the internalization of the Alexa Fluor 555 EGF complex internalization as been reported.

Transferrin Conjugates

Transferrin is a monomeric serum glycoprotein (MW ~80,000 daltons) that binds up to two Fe³⁺ atoms for delivery to vertebrate cells through receptor-mediated endocytosis. Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of the sequestered iron from the transferrin–receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to scavenge more iron. Transferrin uptake is a prototypical and ubiquitous example of clathrin-mediated endocytosis. Although transferrin uptake is widely regarded as a surrogate measure of total clathrin-mediated endocytosis, perturbations that are specific to transferrin endocytosis impel caution in making such extrapolations.

Our fluorescent and biotinylated di-ferric (Fe³⁺) human transferrin conjugates (Transferrin conjugates—Table 16.2) include:

• Fluorescein transferrin (T2871)
• Alexa Fluor 488 transferrin (T13342)
• Alexa Fluor 546 transferrin (T23364)
• Alexa Fluor 555 transferrin (T35352)
• Alexa Fluor 568 transferrin (T23365)
• Alexa Fluor 594 transferrin (T13343, )
• Alexa Fluor 633 transferrin (T23362)
• Alexa Fluor 647 transferrin (T23366)
• Alexa Fluor 680 transferrin (T35357)
• Tetramethylrhodamine transferrin (T2872)
• Texas Red transferrin (T2875)
• Biotin-XX transferrin (T23363) 

Alexa Fluor transferrin conjugates are highly recommended because of their brightness, enhanced photostability and lack of sensitivity to pH (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3). The pH sensitivity of fluorescein-labeled transferrin has been exploited to investigate events occurring during endosomal acidification. Fluorescent transferrins have also been used to:

• Analyze the role of the γ-chain of type III IgG receptors in antigen–antibody complex internalization
• Characterize endocytic apparatus phenotypes in drug-resistant cancer cells
• Demonstrate that the fungal metabolite brefeldin A (B7450, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4) induces an increase in tubulation of transferrin receptors in BHK-21 cells and in the perikaryal–dendritic region of cultured hippocampal neurons
• Image transferrin receptor dynamics using FRET
• Observe receptor trafficking in live cells by confocal laser-scanning microscopy

Uptake of a horseradish peroxidase (HRP) conjugate of transferrin by endosomes has been detected using tyramide signal amplification (TSA, Peroxidase-Based Signal Amplification, Including TSA—Section 6.2) by catalytic deposition of biotin tyramide and use of fluorescent streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).

In addition to fluorescent and biotinylated transferrin conjugates, we offer a mouse monoclonal IgG₁ anti–human transferrin receptor antibody (A11130). This antibody can be used with any of our Zenon Mouse IgG₁ Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7) for rapid preparation of labeling complexes. Antibodies against transferrin receptors have been used for indirect immunofluorescent staining of the transferrin receptor, transport of molecules across the blood–brain barrier, characterization of transferrin in recycling compartments, enzyme-linked immunosorbent assays (ELISAs) and antibody competition with transferrin uptake.

Fluorescent Fibrinogen

Fibrinogen is a key component in the blood clotting process and can support both platelet–platelet and platelet–surface interactions by binding to the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor (also called integrin α_IIbβ₃) of activated platelets. Activation of GPIIb-IIIa is required for fibrinogen binding, which leads to platelet activation, adhesion, spreading and microfilament reorganization of human endothelial cells in vitro. Bone marrow transplant patients have significantly higher levels of fibrinogen binding, as compared with controls Soluble fibrinogen binds to its receptor with a Ca²⁺-dependent apparent K_d of 0.18 µM. This binding is mediated by the tripeptide sequence Arg–Gly–Asp (RGD), found in both fibrinogen and fibronectin.

Fluorescently labeled fibrinogen has proven to be a valuable tool for investigating platelet activation and subsequent fibrinogen binding. Alexa Fluor 647 fibrinogen has been used to identify activated platelets by flow cytometry. The binding of fluorescein fibrinogen to activated platelets has been shown to be saturable and can be inhibited completely by underivatized fibrinogen.

We offer four conjugates of human fibrinogen in three different fluorescent colors:

• Alexa Fluor 488 human fibrinogen conjugate (F13191)
• Oregon Green 488 human fibrinogen conjugate (F7496)
• Alexa Fluor 546 human fibrinogen conjugate (F13192)
• Alexa Fluor 647 human fibrinogen conjugate (F35200)

These highly fluorescent fibrinogen conjugates are useful for investigating platelet activation and subsequent fibrinogen binding using fluorescence microscopy or flow cytometry (Figure 16.1.7).

DQ Ovalbumin: A Probe for Antigen Processing and Presentation

Although antigen processing and presentation have been extensively studied, the exact sequence and detailed pathways for generating antigenic peptides have yet to be elucidated. In general, the immunogenic protein is internalized by a macrophage, denatured, reduced and proteolyzed, and then the resulting peptides associate with MHC class II molecules that are expressed at the cell surface. Ovalbumin is efficiently processed through mannose receptor–mediated endocytosis by antigen-presenting cells and is widely used for studying antigen processing. DQ ovalbumin (D12053), a self-quenched ovalbumin conjugate, is designed specifically for the study of macrophage-mediated antigen processing in flow cytometry and microscopy assays.

Traditionally, fluorescein-labeled bovine serum albumin (FITC-BSA) has been used as a fluorogenic protein antigen for studying the real-time kinetics of antigen processing in live macrophages by flow cytometry, two-photon fluorescence lifetime imaging microscopy (FLIM) and fluorescence polarization. FITC-ovalbumin has been employed to study antigen uptake in HIV-1–infected monocytic cells. The FITC-ovalbumin and FITC-BSA used in these experiments were heavily labeled with fluorescein such that the intact conjugates were relatively nonfluorescent due to self-quenching. Upon denaturation and proteolysis, however, these FITC conjugates became highly fluorescent, allowing researchers to monitor intracellular trafficking and the processing of ovalbumin and BSA in macrophages.

For studies of antigen processing and presentation, DQ ovalbumin offers several advantages when compared with FITC-ovalbumin and FITC-BSA. Like the FITC conjugates, DQ ovalbumin is labeled with our pH-insensitive, green-fluorescent BODIPY FL dye such that the fluorescence is almost completely quenched until the probe is digested by proteases (Figure 16.1.8). Unlike fluorescein, which has greatly reduced fluorescence intensity at acidic pH and is not very photostable, our BODIPY FL dye exhibits bright, relatively photostable and pH-insensitive fluorescence from pH 3 to 9. Furthermore, the intact DQ ovalbumin is more highly quenched than unprocessed FITC-ovalbumin or FITC-BSA at a lower degree of substitution, thereby providing a lower background signal while preserving the protein's antigenic epitopes. Although we offer the green-fluorescent DQ Green BSA and red-fluorescent DQ Red BSA (D12050, D12051; Detecting Peptidases and Proteases—Section 10.4), which are also self-quenched BODIPY FL and BODIPY TR conjugates, we highly recommend DQ ovalbumin (D12053) for studying antigen processing and presentation because ovalbumin is internalized via the mannose receptor–mediated endocytosis pathway and is thus processed more efficiently by antigen-presenting cells than is BSA.

Fluorescent Gelatin

Collagen is a major component of the extracellular matrix and, in vertebrates, constitutes approximately 25% of total protein. This important protein not only serves a structural role, but also is important in cell adhesion and migration. Specific collagen receptors, fibronectin and a number of other proteins involved in cell–cell and cell–surface adhesion have been demonstrated to bind collagen and gelatin (denatured collagen).

We offer highly fluorescent gelatin conjugates for researchers studying collagen-binding proteins and collagen metabolism, as well as gelatinases and collagenases, which are metalloproteins that digest gelatin and collagen. We offer two green-fluorescent gelatin conjugates—fluorescein gelatin and Oregon Green 488 gelatin (G13187, G13186). Fluorescent gelatin conjugates have been shown to be useful for:

• Assessing gelatinase activity in podosomes of mouse dendritic cells
• Localizing surface fibronectin on cultured cells
• Performing in situ gelatinase zymography on canary brain sections
• Studying fibronectin–gelatin interactions in solution using fluorescence polarization (Fluorescence Polarization (FP)—Note 1.5)

We have also developed fluorogenic gelatinase and collagenase substrates—DQ gelatin and DQ collagen (Figure 16.1.8) (D12054, D12060)—that are described in Detecting Peptidases and Proteases—Section 10.4. In addition, we offer fluorescent microspheres coated with collagen, which are described below.

Fluorescent Casein

Real-time imaging of fluorescein-labeled casein (C2990) and FluoSpheres fluorescent microspheres has been used to characterize the endocytic apparatus of the protozoan Giardia lamblia. The EnzChek Protease Assay Kits (E6638, E6639; Detecting Peptidases and Proteases—Section 10.4) provide convenient fluorescence-based assays for protease activity and contain either green-fluorescent BODIPY FL casein or red-fluorescent BODIPY TR-X casein (Figure 16.1.8). BODIPY FL casein and BODIPY TR-X casein have significant utility as nontoxic and pH-insensitive general markers for phagocytic cells in culture. Our RediPlate 96 (R22132) version of the BODIPY TR-X casein substrate (Detecting Peptidases and Proteases—Section 10.4) is ideal for high-throughput screening of potential protease inhibitors.

Fluorescent Chemotactic Peptide

A variety of white blood cells containing the formyl-Met-Leu-Phe (fMLF) receptor respond to bacterial N-formyl peptides by migrating to the site of bacterial invasion and then initiating an activation pathway to control the spread of infection. Activation involves Ca²⁺ mobilization, transient acidification, actin polymerization, phagocytosis and production of oxidative species. We offer the fluorescein conjugate of the hexapeptide formyl-Nle-Leu-Phe-Nle-Tyr-Lys (F1314), which has been extensively employed to investigate the fMLF receptor. The fluorescein-labeled chemotactic peptide has been used to study G-protein coupling and receptor structure, expression, distribution and internalization.

Fluorescent Insulin

We prepare a high-purity, zinc-free fluorescein isothiocyanate conjugate of human insulin (FITC insulin, I13269). Unlike most commercially available preparations, our FITC insulin is purified by HPLC and consists of a singly labeled species of insulin that has been specifically modified at the N-terminus of the B-chain. Because the degree and position of labeling can alter the biological activity of insulin, we have isolated the singly labeled species that has been shown to retain its biological activity in an autophosphorylation assay. Our FITC insulin preparation is useful for imaging insulin and insulin receptor distribution, as well as for conducting insulin-binding assays using microfluidic devices.

Fluorescent Dexamethasone Probe for Glucocorticoid Receptors

The synthetic steroid hormone dexamethasone binds to the glucocorticoid receptor, producing a steroid–receptor complex that then localizes in the nucleus and regulates gene transcription. In hepatoma tissue culture (HTC) cells, tetramethylrhodamine-labeled dexamethasone has been shown to have high affinity for the glucocorticoid receptor in a cell-free system and to induce tyrosine aminotransferase (TAT) expression in whole cells, albeit at a much lower rate than unmodified dexamethasone. This labeled dexamethasone also allowed the first observations of the fluorescent steroid–receptor complex in the HTC cell cytosol. Fluorescein dexamethasone (D1383) should be similarly useful for studying the mechanism of glucocorticoid receptor activation.

Fluorescent Histone H1

The Alexa Fluor 488 conjugate of the lysine-rich calf thymus histone H1 (H13188) is a useful probe for nuclear protein transport assays. Nuclear-to-mitochondrial translocation of histone H1 is indicative of dsDNA strand breaks. Fluorescent histone H1 conjugates can also be used to detect membrane-surface exposure of acidic phospholipids such as phosphatidylserine.

Fluorescent Probes for the Acrosome Reaction

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases and binds to acrosin, an acrosomal serine protease associated with binding of spermatozoa to the zona pellucida. Alexa Fluor 488 dye–labeled trypsin inhibitor from soybean (T23011) is useful for real-time imaging of the acrosome reaction in live spermatozoa. A fluorescent peanut lectin has been combined with ethidium homodimer-1 (EthD-1, E1169; Viability and Cytotoxicity Assay Reagents—Section 15.2) for a combined acrosome reaction assay and vital staining. Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 conjugates of Arachis hypogaea lectin (PNA) (L21409, L32458, L32459, L32460) have similar utility as acrosomal stains.

Fluorescent Gelatin

Collagen is a major component of the extracellular matrix and, in vertebrates, constitutes approximately 25% of total protein. This important protein not only serves a structural role, but also is important in cell adhesion and migration. Specific collagen receptors, fibronectin and a number of other proteins involved in cell–cell and cell–surface adhesion have been demonstrated to bind collagen and gelatin (denatured collagen).

We offer highly fluorescent gelatin conjugates for researchers studying collagen-binding proteins and collagen metabolism, as well as gelatinases and collagenases, which are metalloproteins that digest gelatin and collagen. We offer two green-fluorescent gelatin conjugates—fluorescein gelatin and Oregon Green 488 gelatin (G13187, G13186). Fluorescent gelatin conjugates have been shown to be useful for:

• Assessing gelatinase activity in podosomes of mouse dendritic cells
• Localizing surface fibronectin on cultured cells
• Performing in situ gelatinase zymography on canary brain sections
• Studying fibronectin–gelatin interactions in solution using fluorescence polarization (Fluorescence Polarization (FP)—Note 1.5)

We have also developed fluorogenic gelatinase and collagenase substrates—DQ gelatin and DQ collagen (Figure 16.1.8) (D12054, D12060)—that are described in Detecting Peptidases and Proteases—Section 10.4. In addition, we offer fluorescent microspheres coated with collagen, which are described below.

Fluorescent Casein

Real-time imaging of fluorescein-labeled casein (C2990) and FluoSpheres fluorescent microspheres has been used to characterize the endocytic apparatus of the protozoan Giardia lamblia. The EnzChek Protease Assay Kits (E6638, E6639; Detecting Peptidases and Proteases—Section 10.4) provide convenient fluorescence-based assays for protease activity and contain either green-fluorescent BODIPY FL casein or red-fluorescent BODIPY TR-X casein (Figure 16.1.8). BODIPY FL casein and BODIPY TR-X casein have significant utility as nontoxic and pH-insensitive general markers for phagocytic cells in culture. Our RediPlate 96 (R22132) version of the BODIPY TR-X casein substrate (Detecting Peptidases and Proteases—Section 10.4) is ideal for high-throughput screening of potential protease inhibitors.

Fluorescent Chemotactic Peptide

A variety of white blood cells containing the formyl-Met-Leu-Phe (fMLF) receptor respond to bacterial N-formyl peptides by migrating to the site of bacterial invasion and then initiating an activation pathway to control the spread of infection. Activation involves Ca²⁺ mobilization, transient acidification, actin polymerization, phagocytosis and production of oxidative species. We offer the fluorescein conjugate of the hexapeptide formyl-Nle-Leu-Phe-Nle-Tyr-Lys (F1314), which has been extensively employed to investigate the fMLF receptor. The fluorescein-labeled chemotactic peptide has been used to study G-protein coupling and receptor structure, expression, distribution and internalization.

Fluorescent Insulin

We prepare a high-purity, zinc-free fluorescein isothiocyanate conjugate of human insulin (FITC insulin, I13269). Unlike most commercially available preparations, our FITC insulin is purified by HPLC and consists of a singly labeled species of insulin that has been specifically modified at the N-terminus of the B-chain. Because the degree and position of labeling can alter the biological activity of insulin, we have isolated the singly labeled species that has been shown to retain its biological activity in an autophosphorylation assay. Our FITC insulin preparation is useful for imaging insulin and insulin receptor distribution, as well as for conducting insulin-binding assays using microfluidic devices.

Fluorescent Dexamethasone Probe for Glucocorticoid Receptors

The synthetic steroid hormone dexamethasone binds to the glucocorticoid receptor, producing a steroid–receptor complex that then localizes in the nucleus and regulates gene transcription. In hepatoma tissue culture (HTC) cells, tetramethylrhodamine-labeled dexamethasone has been shown to have high affinity for the glucocorticoid receptor in a cell-free system and to induce tyrosine aminotransferase (TAT) expression in whole cells, albeit at a much lower rate than unmodified dexamethasone. This labeled dexamethasone also allowed the first observations of the fluorescent steroid–receptor complex in the HTC cell cytosol. Fluorescein dexamethasone (D1383) should be similarly useful for studying the mechanism of glucocorticoid receptor activation.

Fluorescent Histone H1

The Alexa Fluor 488 conjugate of the lysine-rich calf thymus histone H1 (H13188) is a useful probe for nuclear protein transport assays. Nuclear-to-mitochondrial translocation of histone H1 is indicative of dsDNA strand breaks. Fluorescent histone H1 conjugates can also be used to detect membrane-surface exposure of acidic phospholipids such as phosphatidylserine.

Fluorescent Probes for the Acrosome Reaction

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases and binds to acrosin, an acrosomal serine protease associated with binding of spermatozoa to the zona pellucida. Alexa Fluor 488 dye–labeled trypsin inhibitor from soybean (T23011) is useful for real-time imaging of the acrosome reaction in live spermatozoa. A fluorescent peanut lectin has been combined with ethidium homodimer-1 (EthD-1, E1169; Viability and Cytotoxicity Assay Reagents—Section 15.2) for a combined acrosome reaction assay and vital staining. Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 conjugates of Arachis hypogaea lectin (PNA) (L21409, L32458, L32459, L32460) have similar utility as acrosomal stains.

Amine-Reactive OxyBURST Green Reagent

As an alternative to Fc OxyBURST Green assay reagent and OxyBURST Green H₂HFF BSA, we offer amine-reactive OxyBURST Green H₂DCFDA succinimidyl ester (2',7'-dichlorodihydrofluorescein diacetate, SE; D2935), which can be used to prepare oxidation-sensitive conjugates of a wide variety of biomolecules and particles, including antibodies, antigens, peptides, proteins, dextrans, bacteria, yeast and polystyrene microspheres. Following conjugation to amines, the two acetates of OxyBURST Green H₂DCFDA reagent can be removed by treatment with hydroxylamine at near-neutral pH to yield the oxidant-sensitive dichlorodihydrofluorescein conjugates. Thus, like our Fc OxyBURST Green assay reagent, they provide a means of detecting the oxidative burst in phagocytic cells.

Several other reagents—dihydrofluoresceins, dihydrorhodamines, dihydroethidium and chemiluminescent probes—that have been used to detect the reactive oxygen species (ROS) produced during phagocytosis are described in Generating and Detecting Reactive Oxygen Species—Section 18.2.

Fluorescent Low-Density Lipoprotein Complexes

The human LDL complex, which delivers cholesterol to cells by receptor-mediated endocytosis, comprises a core of about 1500 molecules of cholesteryl ester and triglyceride, surrounded by a 20 Å–thick shell of phospholipids, unesterified cholesterol and a single copy of apoprotein B 100 (MW ~500,000 daltons). Once internalized, LDL dissociates from its receptor and eventually appears in lysosomes. In addition to unlabeled LDL (L3486), which has been reported to be an effective vehicle for selectively delivering antitumor drugs to cancer cells, we offer two classes of labeled LDL probes—those containing an unmodified apoprotein, used to study the mechanisms of normal cholesterol delivery and internalization, and those with an acetylated apoprotein, used to study endothelial, microglial and other cell types that express receptors that specifically bind this modified LDL (see below).

For the class of labeled LDL probes containing unmodified apoprotein, we prepare LDL noncovalently labeled with either DiI (DiI LDL, L3482) or the BODIPY FL fluorophore (BODIPY FL LDL, L3483), highly fluorescent lipophilic dyes that diffuse into the hydrophobic portion of the LDL complex without affecting the LDL-specific binding of the apoprotein. As compared with DiI LDL, BODIPY FL LDL is more efficiently excited by the 488 nm spectral line of the argon-ion laser, making it better suited for flow cytometry and confocal laser-scanning microscopy studies. Like our BODIPY FL C₅-ceramide (D3521, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4), BODIPY FL LDL may exhibit concentration-dependent long-wavelength emission (>550 nm), precluding its use for multicolor labeling with red fluorophores. Both DiI LDL and BODIPY FL LDL have been used to investigate the binding specificity and partitioning of LDL throughout the Schistosoma mansoni parasite (). Fluorescent LDL complexes have also proven useful in a variety of experimental systems to:

• Count the number of cell-surface LDL receptors, analyze their motion and clustering and follow their internalization
• Demonstrate that fibroblasts grown continuously in the presence of DiI LDL (L3482) proliferate normally and exhibit normal morphology, making DiI LDL a valuable alternative to ¹²⁵I-labeled LDL for quantitating LDL receptor activity
• Identify LDL receptor–deficient Chinese hamster ovary (CHO) cell mutants
• Image LDL receptor endocytosis in COS7 cells expressing green-fluorescent protein (GFP)–tagged GTPase
• Investigate the modulation of LDL receptor expression by statin drugs

We prepare fluorescent LDL products from fresh human plasma, and they should be stored refrigerated and protected from light; LDL products must not be frozen. Because preparation of these complexes involves several variables, some batch-to-batch variability in degree of labeling and fluorescence yield is expected.

Fluorescent Acetylated LDL Complexes

If the lysine residues of LDL's apoprotein have been acetylated, the LDL complex no longer binds to the LDL receptor, but rather is taken up by macrophage and endothelial cells that possess "scavenger" receptors specific for the modified LDL. Once the acetylated LDL (AcLDL) complexes accumulate within these cells, they assume an appearance similar to that of foam cells found in atherosclerotic plaques. We offer unlabeled AcLDL (L35354), as well as AcLDL noncovalently labeled with DiI (L3484) and AcLDL covalently labeled with Alexa Fluor 488 dye (L23380), Alexa Fluor 594 dye (L35353) or BODIPY FL dye (L3485). Fluorescent dye conjugates of high-density lipoproteins, including one prepared using Alexa Fluor 488 succinimidyl ester (A20000, A20100; Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), are taken up via the same receptor as acetylated LDL complexes.

Using DiI AcLDL, researchers have discovered that the scavenger receptors on rabbit fibroblasts and smooth muscle cells appear to be up-regulated through activation of the protein kinase C pathway. DiI AcLDL has also been used to show that Chinese hamster ovary (CHO) cells express AcLDL receptors that are distinct from macrophage scavenger receptors. Ultrastructural localization of endocytic compartments that maintain a connection to the extracellular space has been achieved by photoconversion of DiI AcLDL in the presence of diaminobenzidine (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). A quantitative assay for LDL- and scavenger-receptor activity in adherent and nonadherent cultured cells that avoids the use of both radioactivity and organic solvents has been described.

It has now become routine to identify endothelial cells and microglial cells in primary cell culture by their ability to take up DiI AcLDL (). DiI AcLDL was employed in order to confirm endothelial cell identity in investigations of shear stress and P-glycoprotein expression, as well as to identify blood vessels in a growing murine melanoma. In addition, patch-clamp techniques have been used to investigate membrane currents in mouse microglia, which were identified both in culture and in brain slices by their staining with DiI AcLDL. For some applications, Alexa Fluor 488, Alexa Fluor 594 and BODIPY FL AcLDL may be the preferred probes because the dyes are covalently bound to the modified apoprotein portion of the LDL complex and are therefore not extracted during subsequent manipulations of the cells. Furthermore, the green-fluorescent Alexa Fluor 488 AcLDL has spectral characteristics similar to fluorescein and is useful for analyses with instruments equipped with the 488 nm argon-ion laser excitation sources, including flow cytometers and confocal laser-scanning microscopes. The bright and photostable red-fluorescent Alexa Fluor 594 AcLDL conjugate is useful for multilabeling experiments with green-fluorescent probes and can be efficiently excited by the 594 nm spectral line of the orange He-Ne laser.

Fluorescent Lipopolysaccharides

We offer fluorescent conjugates of lipopolysaccharides (LPS) from Escherichia coli and Salmonella minnesota (Fluorescent lipopolysaccharide conjugates—Table 16.1), including:

• Alexa Fluor 488 LPS from E. coli serotype 055:B5 (L23351)
• Alexa Fluor 488 LPS from S. minnesota (L23356)
• Alexa Fluor 568 LPS from E. coli serotype 055:B5 (L23352)
• Alexa Fluor 594 LPS from E. coli serotype 055:B5 (L23353)
• BODIPY FL LPS from E. coli serotype 055:B5 (L23350)

LPS, also known as endotoxins, are a family of complex glycolipid molecules located on the surface of gram-negative bacteria. LPS play a large role in protecting the bacterium from host defense mechanisms and antibiotics. Binding of LPS to the CD14 cell-surface receptor of phagocytes is the key initiation step in the mammalian immune response to infection by gram-negative bacteria. The structural core of LPS, and the primary determinant of its biological activity, is an N-acetylglucosamine derivative, lipid A (Figure 16.1.5). In many gram-negative bacterial infections, LPS are responsible for clinically significant symptoms like fever, low blood pressure and tissue edema, which can lead to disseminated intravascular coagulation, organ failure and death.

The fluorescent BODIPY FL and Alexa Fluor LPS conjugates, which are labeled with succinimidyl esters of these dyes, allow researchers to follow LPS-elicited inflammatory responses. Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes. Alexa Fluor 488 LPS conjugates (L23351, L23356) have been shown to selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes and microglia.

The BODIPY FL derivative of LPS from E. coli strain LCD25 (L23350) was used to measure the transfer rate of LPS from monocytes to high-density lipoprotein (HDL). Another study utilized a BODIPY FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi apparatus in neutrophils, although this could have been due to probe metabolism. It has been reported that organelles other than the Golgi are labeled by some fluorescent or nonfluorescent LPS. Cationic lipids are reported to assist the translocation of fluorescent lipopolysaccharides into live cells; cell surface–bound LPS can be quenched by trypan blue.

Other probes useful for analyzing lipopolysaccharides include fluorescent analogs of the LPS-binding antibiotic polymyxin B (Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3) and BODIPY TR cadaverine (D6251, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4). BODIPY TR cadaverine binds with high selectivity to lipid A, forming the basis for high-throughput ligand displacement assays for identifying endotoxin antagonists.

Many of the fluorescent ligands described in this section first bind to cell-surface receptors, then are internalized and, in some cases, recycled to the cell surface. In most applications, the cell-surface and internalized ligand populations are spatially resolved by imaging. It is often desirable to include noninternalized plasma membrane reference markers in these labeling protocols. CellMask Orange and CellMask Deep Red plasma membrane stains (C10045, C10046; Tracers for Membrane Labeling—Section 14.4) are particularly suitable for this purpose. Other useful membrane markers include post-translationally lipidated fluorescent proteins (O36214, O10139; Tracers for Membrane Labeling—Section 14.4). When spatial resolution is not possible, there are other means by which these signals can be separated and, in some cases, quantitated. These include:

• Use of antibodies to the Alexa Fluor 488, BODIPY FL, fluorescein/Oregon Green, tetramethylrhodamine, Texas Red and Alexa Fluor 405/Cascade Blue dyes (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore antibodies and their conjugates—Table 7.8) to quench most of the fluorescence of surface-bound or exocytosed probes.
• Use of a dye such as trypan blue to quench external fluorescent signals but not internalized signals (Figure 16.1.9)—a method employed in our Vybrant Phagocytosis Assay Kit (V6694) described below.
• Rapid acidification of the medium to quench the fluorescence of pH-sensitive fluorophores such as fluorescein on the cell surface, thus enabling selective detection of endocytosed probe.
• Tagging of proteins, polysaccharides, cells, bacteria, yeast, fungi and other materials to be endocytosed with a pH-sensitive dye—such as our pHrodo, SNARF or Oregon Green dyes (pH Indicators—Chapter 20)—that undergoes a spectral shift or intensity change in the acidic pH range found in phagovacuoles and late endosomes.
• Use of heavily labeled, highly quenched proteins such as our DQ BSA and DQ gelatin probes, which yield highly fluorescent peptides upon intracellular proteolysis (Detecting Peptidases and Proteases—Section 10.4).

Pathway-specific inhibitors—such as chlorpromazine, dynasore (Figure 16.1.15), dansyl cadaverine (D113, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4), brefeldin A (B7450, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4), genistein and filipin—are widely used in combination with fluorescently labeled ligands for characterizing endocytic pathways. A critical evaluation highlights some necessary cautions in the application and interpretation of this approach, relating to decreased cell viability caused by some inhibitors as well as cell-type dependent differences in their efficacy.

FM 1-43

FM dyes—FM 1-43, FM 2-10, FM 4-64, FM 5-95 and the aldehyde-fixable FM 1-43FX and FM 4-64FX—are excellent membrane probes both for identifying actively firing neurons and for investigating the mechanisms of activity-dependent vesicle cycling in widely different species. FM dyes may also be useful as general-purpose probes for investigating endocytosis and for simply identifying cell membrane boundaries.

FM 1-43 and its analogs, which are nontoxic to cells and virtually nonfluorescent in aqueous medium, are believed to insert into the outer leaflet of the surface membrane where they become intensely fluorescent. In a neuron that is actively releasing neurotransmitters, these dyes become internalized within the recycled synaptic vesicles and the nerve terminals become brightly stained (, ). The nonspecific staining of cell-surface membranes can simply be washed off prior to viewing. Wash removal of noninternalized dye background is more difficult in tissue preparations than in disseminated cell cultures. Extracellular fluorescence quenching and dye adsorption strategies have been developed to address this problem. Alternatively, the optical sectioning capabilities of confocal microscopy, two-photon excitation microscopy (Fluorescent Probes for Two-Photon Microscopy—Note 1.5) and total internal reflection (TIRF) microscopy provide instrument-based solutions for improving the signal-to-background contrast. The amount of FM 1-43 taken up per vesicle by endocytosis equals the amount of dye released upon exocytosis, indicating that the dye does not transfer from internalized vesicles to an endosome-like compartment during the recycling process. In astrocytes, internalization of FM 1-43 (and FM 4-64) is mediated by store-operated calcium channels and not by endocytosis. Like most styryl dyes, the absorption and fluorescence emission spectra of FM 1-43 are significantly shifted in the membrane environment and are relatively broad (), requiring careful matching with other fluorophores to avoid channel crosstalk in multiplex detection applications (Using the Fluorescence SpectraViewer—Note 23.1). We offer FM 1-43 in a 1 mg vial (T3163) or specially packaged in 10 vials of 100 µg each (T35356).

FM 1-43 was employed in a study showing that synaptosomal endocytosis is independent of both extracellular Ca²⁺ and membrane potential in dissociated hippocampal neurons, as well as in a spectrofluorometric assay demonstrating that nitric oxide–stimulated vesicle release is independent of Ca²⁺ in isolated rat hippocampal nerve terminals. FM 1-43 has been used in combination with fura-2 (Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2) to simultaneously measure intracellular Ca²⁺ and membrane turnover. FM 1-43 dye–mediated photoconversion has been used to visualize recycling vesicles in hippocampal neurons.

Other Analogs of FM 1-43

A comparison of mammalian motor nerve terminals stained with either FM 1-43 or the more hydrophilic analog FM 2-10 (T7508, ) revealed that lower background staining by FM 2-10 and its faster destaining rate may make it the preferred probe for quantitative applications. However, staining with FM 2-10 requires much higher dye concentrations (100 µM compared with 2 µM for FM 1-43). Additionally, it has been shown that both FM 1-43 and FM 2-10 are antagonists of muscarinic acetylcholine receptors and may be useful for analyzing receptor distribution and occupancy. This property may be due to the cationic alkylammonium substituent of FM dyes, which they have in common with choline, and could serve as one of the sources of background FM dye staining in tissues.

FM 4-64 (T3166, T13320) and RH 414 (T1111)—both more hydrophobic than FM 1-43—may also be useful as probes for investigating endocytosis. Because small differences in the polarity of these FM probes can play a large role in their rates of uptake and their retention properties, we have introduced FM 5-95 (T23360), a slightly less lipophilic analog of FM 4-64 with essentially identical spectroscopic properties. FM 4-64 exhibits long-wavelength red fluorescence that can be distinguished from the green-fluorescent protein (GFP) with the proper optical filter sets.

FM 4-64 is an endosomal marker and vital stain that persists through cell division, as well as a stain for functional presynaptic boutons. In addition, FM 4-64 staining has been used to visualize membrane migration and fusion during Bacillus subtilis sporulation, and these movements can be correlated with the translocation of GFP-labeled proteins (). Sequential pulse-chase application of FM 4-64 and FM 1-43 allows two-color fluorescence discrimination of temporally staged synaptic vesicles populations. FM 4-64 selectively stains yeast vacuolar membranes and is an important tool for visualizing vacuolar organelle morphology and dynamics and for studying the endocytic pathway and vacuole fusion in yeast (Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3). FM 4-64 and FM 1-43 also have many applications for visualizing membrane dynamics in plant and algal cells.

FM 1-43FX and FM 4-64FX: Fixable FM Dyes

FM 1-43FX and FM 4-64FX are FM 1-43 and FM 4-64 analogs, respectively, that have been modified to contain an aliphatic amine. This modification makes the probe fixable with aldehyde-based fixatives, including formaldehyde and glutaraldehyde. FM 1-43FX has been used to study synaptic vesicle cycling in cone photoreceptor terminals and to investigate the functional maturation of glutamatergic synapses. FM 1-43FX (F35355) and FM 4-64FX (F34653) are available specially packaged in 10 vials of 100 µg each.

4-Di-1-ASP and 4-Di-2-ASP

The cationic mitochondrial dyes 4-Di-1-ASP (D288) and 4-Di-2-ASP (D289) stain presynaptic nerve terminals independent of neuronal activity. These aminostyrylpyridinium dyes have also been widely used as substrates for functional analysis of biogenic amine transporters and renal and hepatic organic cation transporters.

TMA-DPH

Also useful as a lipid marker for endocytosis and exocytosis is the cationic linear polyene TMA-DPH (T204), which readily incorporates in the plasma membrane of live cells. TMA-DPH is virtually nonfluorescent in water and is reported to bind to cells in proportion to the available membrane surface. Its fluorescence intensity is therefore sensitive to physiological processes that cause a net change in membrane surface area, making it an excellent probe for monitoring events such as changes in cell volume and exocytosis.

Fluorescent Cholera Toxin Subunit B: Markers of Lipid Rafts

Fluorescent cholera toxins, which bind to galactosyl moieties, are markers of lipid rafts—regions of cell membranes high in ganglioside G_M1 that are thought to be important in cell signaling (Cholera Toxin Subunits A and B—Note 7.5). Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by their selective interactions with these membrane microdomains. Recent research has demonstrated that lipid rafts play a role in a variety of cellular processes—including the compartmentalization of cell-signaling events, the regulation of apoptosis and the intracellular trafficking of certain membrane proteins and lipids —as well as in the infectious cycles of several viruses and bacterial pathogens.

The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405; Tracers for Membrane Labeling—Section 14.4) provide the key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes (, ). Live cells are first labeled with the green-fluorescent Alexa Fluor 488, orange-fluorescent Alexa Fluor 555 or red-fluorescent Alexa Fluor 594 conjugate of cholera toxin subunit B (CT-B). This CT-B conjugate binds to the pentasaccharide chain of plasma membrane ganglioside G_M1, which selectively partitions into lipid rafts. An antibody that specifically recognizes CT-B is then used to crosslink the CT-B–labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by fluorescence microscopy. Each Vybrant Lipid Raft Labeling Kit contains sufficient reagents to label 50 live-cell samples, including:

• Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor 488 (in Kit V34403), Alexa Fluor 555 (in Kit V34404) or Alexa Fluor 594 (in Kit V34405) dye
• Anti–cholera toxin subunit B antibody (anti–CT-B)
• Concentrated phosphate-buffered saline (PBS)
• Detailed labeling protocol (Vybrant Lipid Raft Labeling Kits)

Cholera toxin subunit B and its conjugates are also established as superior tracers for retrograde labeling of neurons. Cholera toxin subunit B conjugates bind to the pentasaccharide chain of ganglioside G_M1 on neuronal cell surfaces and are actively taken up and transported; alternatively, they can be injected by iontophoresis. Unlike the carbocyanine-based neuronal tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4), cholera toxin subunit B conjugates can be used on tissue sections that will be fixed and frozen.

All of our cholera toxin subunit B conjugates are prepared from recombinant cholera toxin subunit B, which is completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. The Alexa Fluor 488 (C22841, C34775), Alexa Fluor 555 (C22843, C34776), Alexa Fluor 594 (C22842, C34777) and Alexa Fluor 647 (C34778) conjugates of cholera toxin subunit B combine this versatile tracer with the superior brightness of our Alexa Fluor dyes to provide sensitive and selective receptor labeling and neuronal tracing. We also offer biotin-XX (C34779) and horseradish peroxidase (C34780) conjugates of cholera toxin subunit B for use in combination with diaminobenzidine (DAB) oxidation, tyramide signal amplification (TSA) and Qdot nanocrystal–streptavidin conjugates.

Fluorescent Protein–Based Lipid Raft Markers

CellLight plasma membrane expression vectors (C10606, C10607, C10608; Tracers for Membrane Labeling—Section 14.4) generate cyan-, green- or red-autofluorescent proteins fused to a plasma membrane targeting sequence consisting of the 10 N-terminal amino acids of Lck tyrosine kinase (Lck10). These fusion proteins are lipid raft markers with well established utility, providing alternatives to cholera toxin B conjugates or BODIPY FL C₅-ganglioside G_M1 (B13950, B34401; Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3) with the inherent advantages of long-lasting and titratable expression conferred by BacMam 2.0 vector technology (BacMam Gene Delivery and Expression Technology—Note 11.1).

Fluorescent Protein–Based Synaptic Vesicle Marker

CellLight Synaptophysin-RFP (C10610) is a valuable counterpart to FM dyes for visualizing the distribution and density of presynaptic sites in neurons both in vitro and in vivo. Synaptophysin is a synaptic vesicle membrane glycoprotein that is involved in the biogenesis and fusion of synaptic vesicles but is not essential for neurotransmitter release. It is found in virtually all synaptically active neurons in the brain and spinal cord. This CellLight reagent incorporates all the customary advantages of BacMam 2.0 delivery technology including high transduction efficiency and long-lasting and titratable expression (BacMam Gene Delivery and Expression Technology—Note 11.1).

Anti–Synapsin I Antibody

Synapsin I is an actin-binding protein that is localized exclusively to synaptic vesicles and thus serves as an reliable marker for synapses in brain and other neuronal tissues. Synapsin I inhibits neurotransmitter release, an effect that is abolished upon its phosphorylation by Ca²⁺/calmodulin–dependent protein kinase II (CaM kinase II). Antibodies directed against synapsin I have proven valuable in molecular and neurobiology research, for example, to estimate synaptic density and to follow synaptogenesis.

We offer a rabbit polyclonal anti–bovine synapsin I antibody as an affinity-purified IgG fraction (A6442). This antibody was isolated from rabbits immunized against bovine brain synapsin I but is also active against human, rat and mouse forms of the antigen; it has little or no activity against synapsin II. The affinity-purified rabbit polyclonal antibody was fractionated from the serum using column chromatography in which bovine synapsin I was covalently bound to the column matrix. Affinity-purified anti–synapsin I antibody is suitable for immunohistochemistry (), western blots, enzyme-linked immunoadsorbent assays and immunoprecipitations. Our complete selection of antibodies can be found at www.invitrogen.com/handbook/antibodies.

Fluorescent Protein–Based Endosomal Markers

CellLight Early Endosomes–GFP (C10586) and CellLight Early Endosomes–RFP (C10587) reagents provide BacMam expression vectors encoding fusions of GFP or RFP with the small GTPase Rab5a. Rab5a fusions with autofluorescent proteins are sensitive and precise early endosome markers for real-time imaging of clathrin-mediated endocytosis in live cells. We also offer CellLight Late Endosomes–GFP (C10588) and CellLight Late Endosomes–RFP (C10589) reagents, which are BacMam expression vectors encoding fusions of GFP or RFP with the late-endosomal protein Rab7a. These CellLight reagents incorporate all the customary advantages of BacMam 2.0 delivery technology including high transduction efficiency and long-lasting and titratable expression (BacMam Gene Delivery and Expression Technology—Note 11.1).

BioParticles Fluorescent Bacteria and Yeast

The BioParticles product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes and natural antigenicities. These fluorescent BioParticles products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry and flow cytometry. We offer Escherichia coli (K-12 strain), Staphylococcus aureus (Wood strain without protein A) and zymosan (Saccharomyces cerevisiae) BioParticles products covalently labeled with a variety of fluorophores, including Alexa Fluor, fluorescein, BODIPY FL, tetramethylrhodamine, Texas Red and pHrodo dyes (BioParticles fluorescent bacteria and yeast—Table 16.3). Special care has been taken to remove any free dye after conjugation. BioParticles products are freeze-dried and ready for reconstitution in a buffer of choice and are supplied with a general protocol for measuring phagocytosis; we also offer opsonizing reagents for use with each particle type, as described below.

Unlike the fluorescence of fluorescein-labeled BioParticles bacteria and yeast, which is strongly quenched in acidic environments, the fluorescence of the Alexa Fluor 488, BODIPY FL, tetramethylrhodamine and Texas Red BioParticles conjugates is uniformly intense between pH 3 and 10. This property is particularly useful for quantitating fluorescent bacteria and zymosan within acidic phagocytic vacuoles.

Fluorescent bacteria and yeast particles are proven tools for studying a variety of phagocytosis parameters. For example, they have been used to:

• Detect the phagocytosis of yeast by murine peritoneal macrophage and human neutrophils
• Determine the effects of different opsonization procedures on the efficiency of phagocytosis of pathogenic bacteria and yeast
• Investigate the kinetics of phagocytosis degranulation and actin polymerization in stimulated leukocytes
• Quantitate the effects of purinergic P2X₇ receptor activation on phagosomal maturation
• Show that Dictyostelium discoideum depleted of clathrin heavy chains are still able to undergo phagocytosis of fluorescent zymosans
• Study molecular defects in phagocytic function
  

Vybrant Phagocytosis Assay Kit

The Vybrant Phagocytosis Assay Kit (V6694) provides a convenient set of reagents for quantitating phagocytosis and assessing the effects of certain drugs or conditions on this cellular process. In this assay, cells of interest are incubated first with green-fluorescent fluorescein-labeled E. coli BioParticles conjugates, which are internalized by phagocytosis, and then with trypan blue, which quenches the fluorescence of any extracellular BioParticles product (Figure 16.1.9). The methodology provided by this kit was developed using the adherent murine macrophage cell line J774; however, researchers have adapted this assay to other phagocytic cell types and other instrument platforms such as flow cytometers. Each kit provides sufficient reagents for 250 tests using a 96-well microplate format and contains:

• BioParticles fluorescein-labeled Escherichia coli
• Hanks' balanced salt solution (HBSS)
• Trypan blue
• Step-by-step instructions for performing the phagocytosis assay (Vybrant Phagocytosis Assay Kit)

pHrodo BioParticles Fluorescent Bacteria

In contrast to both the fluorescein- and Alexa Fluor dye–labeled BioParticles conjugates, the fluorescence of the pHrodo E. coli and S. aureus BioParticles conjugates (P35361, A10010) increases in acidic environments (Figure 16.1.14), providing a continuous positive indicator of phagocytic uptake. With a simple no-cell background subtraction method, a large and specific signal is obtained from cells that ingest the pHrodo BioParticles conjugates, providing a specific index of phagocytosis in the context of a variety of pretreatments or conditions (Figure 16.1.10). The optimal absorption and fluorescence emission maxima of the pHrodo BioParticles conjugates are approximately 560 nm and 585 nm, respectively, but the pHrodo fluorophore is also readily excited by the 488 nm spectral line of the argon-ion laser used in most flow cytometers.

With each pHrodo BioParticles conjugate, we provide sufficient reagent for 100 microplate wells in a 96-well format, along with step-by-step instructions for performing phagocytosis assays in a fluorescence microplate reader. This methodology has been developed using adherent J774A.1 murine macrophage cells, but can be adapted for use with other adherent cells, primary cells  or cells in suspension, as well as for in vivo applications. Cells assayed for phagocytic activity with pHrodo BioParticles conjugates may be fixed with standard formaldehyde solutions for later analysis, preserving differences in signal between control and experimental samples with high fidelity. pHrodo BioParticles conjugate preparations are also amenable to opsonization (E2870, S2860; see below), which can greatly enhance their uptake and signal strength in the phagocytosis assay.

To facilitate the use of pHrodo BioParticles conjugates for the study of phagocytosis, we offer the pHrodo E. coli BioParticles Phagocytosis Kit for Flow Cytometry (A10025), which provides the key reagents for assessing particle ingestion and red blood cell lysis (Figure 16.1.11). Each kit provides sufficient reagents for performing 100 assays when using sample volumes of 100 µL whole blood per assay, including:

• pHrodo E. coli BioParticles conjugates
• Lysis and Wash Buffers
• Detailed protocols (pHrodo E. coli BioParticles Phagocytosis Kit for Flow Cytometry)

pHrodo Phagocytosis Particle Labeling Kit

In addition to the pHrodo BioParticles conjugates, we offer the pHrodo Phagocytosis Particle Labeling Kit for Flow Cytometry (A10026), which allows rapid labeling of biological particles, such as bacteria, and subsequent assessment of of phagocytic activity in whole blood samples by flow cytometry. Each kit provides sufficient reagents for performing 100 assays when using sample volumes of 100 µL whole blood per assay, including:

• pHrodo succinimidyl ester
• Lysis and Wash Buffers
• Dimethylsulfoxide (DMSO)
• Sodium bicarbonate
• Detailed protocols (pHrodo Phagocytosis Particle Labeling Kit for Flow Cytometry)

The amine-reactive pHrodo succinimidyl ester is also available separately (P36600, pH Indicator Conjugates—Section 20.4) for creating pH-sensitive conjugates for following phagocytosis. pHrodo succinimidyl ester was used to label dexamethasone-treated thymocytes for flow cytometry detection of phagocytosis by splenic or peritoneal macrophages.

Opsonizing Reagents and Nonfluorescent BioParticles Products

Many researchers may want to use autologous serum to opsonize their fluorescent zymosan and bacterial particles; however, we also offer special opsonizing reagents (E2870, S2860, Z2850) for enhancing the uptake of each type of particle, along with a protocol for opsonization. These reagents are derived from purified rabbit polyclonal IgG antibodies that are specific for the E. coli, S. aureus or zymosan particles. Reconstitution of the lyophilized opsonizing reagents requires only the addition of water, and one unit of opsonizing reagent is sufficient to opsonize ~10 mg of the corresponding BioParticles product.

In addition, we offer nonfluorescent zymosan (Z2849) and S. aureus (S2859) BioParticles products. These nonfluorescent BioParticles products are useful either as controls or for custom labeling with the reactive dye or indicator of interest.

Fluorescent Polystyrene Microspheres

Fluorescent polystyrene microspheres with diameters between 0.5 and 2.0 µm have been used to investigate phagocytic processes in murine melanoma cells, human alveolar macrophages, ciliated protozoa  and Dictyostelium discoideum. The phagocytosis of fluorescent microspheres has been quantitated both with image analysis  and with flow cytometry. Microspheres—Section 6.5 includes a detailed description of our full line of FluoSpheres (Summary of FluoSpheres fluorescent microspheres—Table 6.7) and TransFluoSpheres (Summary of TransFluoSpheres fluorescent microspheres—Table 6.9) fluorescent microspheres. Because of their low nonspecific binding, carboxylate-modified microspheres appear to be best for phagocytosis applications. For phagocytosis experiments involving multicolor detection, we particularly recommend our 1.0 µm TransFluoSpheres fluorescent microspheres  (T8880, T8883; Microspheres—Section 6.5). Various opsonizing reagents, such as rabbit serum or fetal calf serum, have been used with the microspheres to facilitate phagocytosis.

Fluorescent Microspheres Coated with Collagen

Fibroblasts phagocytose and subsequently digest collagen. These activities play an important role in the remodeling of the extracellular matrix during normal physiological turnover of connective tissues and wound repair, as well as in development and aging. A well-established procedure for observing collagen phagocytosis by either flow cytometry or fluorescence microscopy entails the use of collagen-coated fluorescent microspheres that attach to the cell surface and become engulfed by fibroblasts. We offer yellow-green–fluorescent FluoSpheres collagen I–labeled microspheres in either 1.0 µm or 2.0 µm diameters (F20892, F20893) for use in these applications. In the production of these microspheres, collagen I from calf skin is attached covalently to the microsphere's surface.

Fluorescent Dextrans

Tracing internalization of extracellularly introduced fluorescent dextrans is a standard method for analyzing fluid-phase endocytosis. We offer dextrans with nominal molecular weights ranging from 3000 to 2,000,000 daltons, many of which can also be used as pinocytosis or phagocytosis markers (see Fluorescent and Biotinylated Dextrans—Section 14.5 and Molecular Probes dextran conjugates—Table 14.4 for further discussion and a complete product list). Discrimination of internalized fluorescent dextrans from dextrans in the growth medium is facilitated by use of reagents that quench the fluorescence of the external probe. For example, most of our anti-fluorophore antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore antibodies and their conjugates—Table 7.8) strongly quench the fluorescence of the corresponding dyes.

Negative staining produced by fluorescent dextrans that have been intracellularly infused via a patch pipette is indicative of nonendocytic vacuoles in live pancreatic acinar cells. Extracellular addition of a second, color-contrasting dextran then allows discrimination of endocytic and nonendocytic vacuoles. Intracellular fusion of endosomes has been monitored with a BODIPY FL avidin conjugate by following the fluorescence enhancement that occurs when it complexes with a biotinylated dextran. We have found our Oregon Green 514 streptavidin (S6369, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) to have an over 15-fold increase in fluorescence intensity upon binding free biotin, which may make it the preferred probe for this application (Figure 16.1.12).

pH Indicator Dextrans

The fluorescein dextrans (pKa ~6.4) are frequently used to investigate endocytic acidification. Fluorescence of fluorescein-labeled dextrans is strongly quenched upon acidification; however, fluorescein's lack of a spectral shift in acidic solution makes it difficult to discriminate between an internalized probe that is quenched and residual fluorescence of the external medium. Dextran conjugates that either shift their emission spectra in acidic environments, such as the SNARF dextrans (pH Indicator Conjugates—Section 20.4), or undergo significant shifts of their excitation spectra, such as BCECF and Oregon Green dextrans (pH Indicator Conjugates—Section 20.4), provide alternatives to fluorescein. The Oregon Green 488 and Oregon Green 514 dextrans exhibit a pKa of approximately 4.7, facilitating measurements in acidic environments. In addition to these pH indicator dextrans, we prepare a dextran that is double-labeled with fluorescein and tetramethylrhodamine (D1951; pH Indicator Conjugates—Section 20.4), which has been used as a ratiometric indicator (Figure 16.1.13) to measure endosomal acidification in Hep G2 cells  and murine alveolar macrophages.

In contrast to fluorescein and Oregon Green 488 dextrans, pHrodo 10,000 MW dextran (P10361) exhibits increasing fluorescence in response to acidification  (Figure 16.1.14). The minimal fluorescent signal from pHrodo dextran at neutral pH prevents the detection of noninternalized and nonspecifically bound conjugates and eliminates the need for quenching reagents and extra wash steps, thus providing a simple fluorescent assay for endocytic activity. pHrodo dextran’s excitation and emission maxima of 560 and 585 nm, respectively, facilitate multiplexing with other fluorophores including blue-, green- and far-red–fluorescent probes. Although pHrodo dextran is optimally excited at approximately 560 nm, it is also readily excited by the 488 nm spectral line of the argon-ion laser found on flow cytometers, confocal microscopes and imaging microplate readers (Figure 16.1.15).

Hydrophilic fluorescent dyes—including sulforhodamine 101 (S359), lucifer yellow CH (L453), calcein (C481), 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, pyranine; H348) and Cascade Blue hydrazide (C687)—are taken up by actively firing neurons through endocytic recycling of the synaptic vesicles. Unlike the fluorescent FM membrane probes described above, however, the hydrophilic fluorophores appear to work for only a limited number of species in this application. In some tissue preparations, background due to noninternalized polar markers is easier to wash away than that emanating from membrane markers such as FM 1-43. The same dyes have frequently been used as fluid-phase markers of pinocytosis. The highly water-soluble Alexa Fluor hydrazides and Alexa Fluor hydroxylamines (Polar Tracers—Section 14.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2) provide superior spectral properties and can be fixed in cells by aldehyde-based fixatives.