Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4

Page Contents

• Cell Enumeration and Cell Proliferation Assays for Animal Cells
• Detection and Enumeration Assays for Microorganisms and Viruses
• Countess Automated Cell Counter
• Premo FUCCI Cell Cycle Sensor
• Nucleic Acid Probes for Cell-Cycle Analysis
• HCS Mitotic Index Kit
• Data Table
• Ordering Information

Cell proliferation and the characterization of agents that either promote or retard cell proliferation are extremely important areas of cell biology and drug-discovery research. We offer both traditional reagents for assessing cell proliferation and cell cycle—in particular the Hoechst nucleic acid stains and probes for 5-bromo-2'-deoxyuridine (BrdU) incorporation during cell division—as well as some exceptional tools developed in our laboratories, including the Click-iT EdU cell proliferation assay. For simply detecting the presence or counting the number of cells, fluorescent stains that identify cells by their characteristic morphology or light-scattering properties may be sufficient.

Reagents for counting cells and quantitating cell proliferation are valuable research tools. Most cell proliferation assays estimate the number of cells either by incorporating a modified nucleotide into cells during proliferation or by measuring total nucleic acid or protein content of lysed cells. Several of our nucleic acid stains (Nucleic Acid Stains—Section 8.1) and nucleotides (Labeling Oligonucleotides and Nucleic Acids—Section 8.2) have proven useful in nucleic acid labeling protocols. Here we describe our Click-iT EdU cell proliferation assay, which provides a superior alternative to bromodeoxyuridine (BrdU) or ³H-thymidine incorporation methods for measuring new DNA synthesis. Alternatively, our CyQUANT Cell Proliferation Assay Kits use the CyQUANT GR, CyQUANT NF or CyQUANT Direct nucleic acid stains to measure increases in nucleic acid content that accompany cell proliferation.

Click-iT Tools for Cell Proliferation Analysis

Click-iT labeling technology employs a bioorthogonal reactive chemistry for the in situ labeling of specific molecular populations, such as newly synthesized nucleic acids, in an experimental time window of interest. The Click-iT labeling reaction is based on a copper-catalyzed azide–alkyne cycloaddition and derives its high degree of specificity from the fact that the azide and alkyne reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms. Click-iT labeling technology and the details of the click reaction are discussed in Click Chemistry—Section 3.1. For a complete list azide and alkyne derivatives compatible with Click-iT labeling technology, see Molecular Probes azide and alkyne derivatives—Table 3.1. Here we highlight the Click-iT EdU cell proliferation assay.

The Click-iT EdU cell proliferation assay provides a superior alternative to bromodeoxyuridine (BrdU) or ³H-thymidine incorporation methods for measuring new DNA synthesis. The alkynyl nucleoside analog EdU (5-ethynyl-2'-deoxyuridine; A10044, E10187, E10415) is incorporated into DNA during the synthesis phase (S phase) of the cell cycle and is subsequently detected by copper (I)–catalyzed click coupling to an azide-derivatized fluorophore (Figure 15.4.1). The small size of the click-coupled fluorophore compared to that of antibodies required for immunodetection of BrdU enables efficient penetration of complex samples without the need for harsh cell treatment, simplifying the assay considerably. The Click-iT EdU assay protocol is compatible with both adherent cells and cell suspensions. From start to finish, the EdU detection assay is complete in as little as 90 minutes, as compared with the antibody-based BrdU method, which takes 6–24 hours to complete. In addition, the Click-iT EdU cell proliferation assay can be multiplexed with surface and intracellular marker detection using Alexa Fluor dye–labeled secondary antibodies (Secondary Immunoreagents—Section 7.2) (Figure 15.4.2). Although the majority of applications are in cultured mammalian cells, Click-iT EdU reagents and methods have also been successfully applied to a wide range of model organisms including:

• Escherichia coli
• Caenorhabditis elegans
• Drosophila
• Zebrafish
• Mouse
• Plants (alfalfa, Arabidopsis, grape, maize, rice and tobacco) 

The Click-iT EdU Flow Cytometry Assay Kits provide all the reagents needed to perform 50 assays using 0.5 mL reaction buffer per assay, including the nucleoside analog EdU and all components for fixation, permeabilization and labeling whole blood samples, adherent cells or suspension cells. We offer three Click-iT EdU Flow Cytometry Assay Kits:

• Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (50-assay size, C10425; 100-assay size, C10420)
• Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (50-assay size, C10424; 100-assay size, C10419)
• Click-iT EdU Pacific Blue Flow Cytometry Assay Kit (50-assay size, C10418)

The Click-iT EdU Imaging Kits contain all of the components needed to label and detect incorporated EdU on 50 coverslips using 0.5 mL reaction buffer per test, as well as the blue-fluorescent Hoechst 33342 nuclear stain for performing cell-cycle analysis on adherent cell samples. We offer four Click-iT EdU Imaging Kits:

• Click-iT EdU Alexa Fluor 488 Imaging Kit (C10337)
• Click-iT EdU Alexa Fluor 555 Imaging Kit (C10338)
• Click-iT EdU Alexa Fluor 594 Imaging Kit (C10339)
• Click-iT EdU Alexa Fluor 647 Imaging Kit (C10340)

The Click-iT EdU HCS Assay Kits contain all of the materials needed to label and detect incorporated EdU in adherent cells in 96-well microplates and 100 µL reaction buffer per assay. For cell registration or DNA profiling, these kits also include the blue-fluorescent HCS NuclearMask Blue stain (H10325, Probes for the Nucleus—Section 12.5). We offer four Click-iT EdU HCS Assay Kits:

• Click-iT EdU Alexa Fluor 488 HCS Assay Kit (2-plate size, C10350; 10-plate size, C10351)
• Click-iT EdU Alexa Fluor 555 HCS Assay Kit (2-plate size, C10352; 10-plate size, C10353)
• Click-iT EdU Alexa Fluor 594 HCS Assay Kit (2-plate size, C10354; 10-plate size, C10355)
• Click-iT EdU Alexa Fluor 647 HCS Assay Kit (2-plate size, C10356; 10-plate size, C10357; Figure 15.4.2)

In addition to these kits, our Click-iT EdU Microplate Assay Kit (C10214) provides a simple and rapid workflow with fewer wash steps resulting in a substantial time-savings advantage over traditional BrdU colorimetric or fluorescent cell proliferation assays. This assay uses Oregon Green 488 azide for click coupling to synthetically incorporated EdU. The signal is amplified using immunodetection of the Oregon Green 488 fluorophore by a rabbit anti–Oregon Green horseradish peroxidase (HRP) conjugate followed by fluorogenic or chromogenic detection with our Amplex UltraRed HRP substrate. The Click-iT EdU microplate assay has been successfully tested in HeLa, A549, U2OS and A541 cells with a variety of reagents that modulate DNA synthesis, including the DNA synthesis inhibitor aphidicolin and the mitotic inhibitor paclitaxel. The Click-iT EdU Microplate Assay Kit contains sufficient reagents for performing 400 individual assays in a 96-well plate format.

Figure 15.4.1 Click-iT copper-catalyzed azide–alkyne cycloaddition chemistry applied to detection of newly synthesized DNA. The reaction partners in this example are 5-ethynyl-2'-deoxyuridine (EdU), which can be enzymatically incorporated in DNA during S phase, and the green-fluorescent Alexa Fluor 488 azide.

Figure 15.4.2 Multicolor imaging with the Click-iT EdU Imaging Kits. Muntjac cells were treated with 10 µM EdU for 45 minutes. Cells were then fixed and permeabilized, and EdU that had been incorporated into newly synthesized DNA was detected by the far-red–fluorescent Click-iT EdU Alexa Fluor 647 HCS Assay Kit (C10356, C10357). Tubulin was labeled with an anti-tubulin antibody and visualized with an Alexa Fluor 350 goat anti–mouse IgG antibody (A21049). The Golgi complex was stained with the green-fluorescent Alexa Fluor 488 conjugate of lectin HPA from Helix pomatia (edible snail) (L11271), and peroxisomes were labeled with an anti-peroxisome antibody and visualized with an orange-fluorescent Alexa Fluor 555 donkey anti–rabbit IgG antibody (A31572).

Proliferation Assays Using Bromodeoxyuridine Incorporation

Incorporation of 5-bromo-2'-deoxyuridine (BrdU, B23151) into newly synthesized DNA permits indirect detection of rapidly proliferating cells with fluorescently labeled anti-BrdU antibodies or certain nucleic acid stains, thereby facilitating the identification of cells that have progressed through the S phase of the cell cycle during the BrdU labeling period. We offer fluorescent conjugates of the mouse monoclonal anti-BrdU antibody clone MoBU-1 labeled with our brightest and most photostable dyes:

• Alexa Fluor 488 anti-BrdU antibody (B35130, B35139)
• Alexa Fluor 555 anti-BrdU antibody (B35131)
• Alexa Fluor 594 anti-BrdU antibody (B35132)
• Alexa Fluor 647 anti-BrdU antibody (B35133, B35140)
• Pacific Blue anti-BrdU antibody (B35129)

This anti-BrdU antibody is also available biotinylated (B35138), as well as unlabeled (B35128, B35141). The unlabeled mouse anti-BrdU can be detected with our anti-mouse secondary antibodies (Summary of Molecular Probes secondary antibody conjugates—Table 7.1) using either flow cytometry (Figure 15.4.3) or imaging.

Because fluorescence of the Hoechst 33258 (H1398, H3569, H21491) and Hoechst 33342 (H1399, H3570, H21492) dyes bound to DNA is quenched at sites where BrdU is incorporated, Hoechst dye fluorescence can also be used to detect BrdU incorporation in single cells. This technique has been employed to quantitate the noncycling cell fraction, as well as the fraction of cells that are in G₁ and G₂ of two subsequent cycles. The addition of ethidium bromide as a counterstain that is insensitive to BrdU incorporation allows the resolution of G₁, S and G₂ compartments of up to three consecutive cell cycles.

Unlike the fluorescence of Hoechst dyes, the fluorescence of TO-PRO-3 (T3605) and LDS 751 (L7595) is considerably enhanced by the presence of bromodeoxyuridine in DNA. In conjunction with propidium iodide (P1304MP, P3566, P21493; Nucleic Acid Stains—Section 8.1), these nucleic acid stains have been used to discriminate BrdU-labeled cells from nonproliferating cells by flow cytometry and with an imaging system for automated cell proliferation.

Figure 15.4.3 Detection of proliferation in Wil2S Lymphoma B cells. Cells were treated with 10 µM 5-bromo-2'-deoxyuridine (BrdU, B23151) in culture medium for one hour, then pelleted and fixed with cold 70% ethanol. After treatment with RNase and 4 M HCl (to denature the DNA), the cells were labeled with anti-BrdU antibody and detected using green-fluorescent Alexa Fluor 488 goat anti–mouse IgG antibody (A11001). In addition, the cells were labeled with red-fluorescent propidium iodide (P1304MP, P3566, P21493) to assess the total cellular DNA content. The cells were analyzed by flow cytometry using 488 nm excitation; the fluorescent signals were collected at ~525 nm for the Alexa Fluor 488 dye and at ~675 nm for propidium iodide. Increased BrdU incorporation is indicative of actively proliferating cells.

Proliferation Assays Using ChromaTide Nucleotides

In the strand break induction by photolysis (SBIP) technique, proliferating cells that have incorporated BrdU into newly synthesized DNA are subjected to Hoechst 33258 staining, followed by UV photolysis to induce DNA strand breaks (Figure 15.4.4). Once the cells are fixed, strand breaks can be detected in situ using mammalian terminal deoxynucleotidyl transferase (TdT), which covalently adds labeled nucleotides to the 3'-hydroxyl ends of these DNA fragments. Break sites have traditionally been labeled with biotinylated or haptenylated dUTP conjugates (Labeling Oligonucleotides and Nucleic Acids—Section 8.2) in conjunction with antibodies to the hapten (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) or conjugates of streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). However, a single-step procedure has been described that uses our ChromaTide BODIPY FL-14-dUTP (C7614) as a TdT substrate for directly detecting DNA strand breaks both in BrdU-labeled cells following SBIP and in apoptotic cells (Assays for Apoptosis—Section 15.5; , ).

The single-step BODIPY FL dye–based assay has several advantages over indirect detection of biotinylated or haptenylated nucleotides. With direct detection procedures, no secondary detection reagents are required; fewer protocol steps translate into less chance for error and more immediate results. Moreover, the yield of cells with direct detection procedures is reported to be about three times greater than that of multistep procedures employing biotin- or digoxigenin-conjugated dUTP. Although both BODIPY FL dye– and fluorescein-labeled nucleotides can be detected with fluorescence microscopy or flow cytometry, the BODIPY FL dye–labeled nucleotides provide ~40% stronger signal than fluorescein-labeled nucleotides when assaying strand breaks in apoptotic versus nonapoptotic cells. In addition, fading of the fluorescence of the incorporated BODIPY FL dUTP is less than that of the corresponding fluorescein dUTP analog. Unlike traditional proliferation assays based on BrdU incorporation, no DNA heat- or acid-denaturation steps are required with SBIP in order to visualize the labeled strand breaks, allowing simultaneous detection of the morphology of nuclear proteins and other cellular constituents by immunocytochemical analysis. The narrow emission spectrum of the BODIPY FL dye–labeled nucleotides is especially useful for multicolor labeling experiments.

An elegant technique permits tracking of labeled chromosomes through mitosis by metabolic incorporation of microinjected fluorescent nucleotides, including our fluorescein-12-dUTP (C7604, Labeling Oligonucleotides and Nucleic Acids—Section 8.2), by endogenous cellular enzymes into DNA. The procedure does not interfere with subsequent progress through the cell cycle, and fluorescent strands of DNA can be followed as they assemble into chromosomes and segregate into daughters and granddaughters. Presumably, injection of 5'-bromo-2'-deoxyuridine triphosphate (BrdUTP, B21550), followed by detection of the incorporated BrdU with one of our Alexa Fluor conjugates of anti-BrdU would also be suitable for studying mitosis. The corresponding ribonucleotide (BrUTP, B21551) that has been microinjected into cells is incorporated into RNA of a nucleolar compartment, a process that should also be detectable with fluorescent anti-BrdU conjugates.

Figure 15.4.4 Schematic diagram showing the sequence of events in the strand break induction by photolysis (SBIP) technique. A) Proliferating cells that have incorporated BrdU (*) into newly synthesized DNA are B) exposed to UV light in order to induce DNA strand breaks. If the cells are stained with Hoechst 33258 prior to UV illumination, the photolysis efficiency is increased. C) Once the cells are fixed, the 3'-hydroxyl ends exposed at these strand breaks can be directly labeled in situ using mammalian terminal deoxynucleotidyl transferase (TdT) and our ChromaTide BODIPY FL-14-dUTP (C7614).

Proliferation Assay Using the Succinimidyl Ester of Carboxyfluorescein Diacetate and Related Probes

The succinimidyl ester of carboxyfluorescein diacetate (5(6)-CFDA, SE or CFSE, C1157) is currently the most widely used probe for generation analysis of cells, although our succinimidyl ester of Oregon Green 488 carboxylic acid diacetate (O34550, C34555; see below) offers several important advantages over this fluorescein derivative. CFDA SE spontaneously and irreversibly couples to both intracellular and cell-surface proteins by reaction with lysine side chains and other available amine groups. When cells divide, CFDA SE labeling is distributed equally between the daughter cells, which are therefore half as fluorescent as the parents. As a result, each successive generation in a population of proliferating cells is marked by a halving of cellular fluorescence intensity (excitation/emission maxima ~495/525 nm) that is readily detected by a flow cytometer (Figure 15.4.5), fluorescence microscope or fluorescence microplate reader. CFDA SE is available as a single vial containing 25 mg (C1157). CFDA SE is also available conveniently packaged for cell tracing applications in our Vybrant CFDA SE Cell Tracer Kit (V12883, Figure 15.4.5) and for cell proliferation studies in our CellTrace Cell Proliferation Kit (C34554, Figure 15.4.6). The fluorescent CFDA SE product has excitation/emission maxima of ~492/517 nm and can be detected using a fluorescence microscope, flow cytometer or fluorescence microplate reader. Each kit includes 10 single-use vials of CFDA SE (500 µg each in Kit V12883, 50 µg each in Kit C34554), as well as high-quality anhydrous DMSO and a complete protocol.

CFDA SE produces more homogenous cellular labeling and, consequently, much better intergenerational resolution than other cell-tracking dyes, such as the membrane marker PKH26. Using flow cytometric analysis of CFDA SE labeling, researchers can reliably resolve 8 to 10 successive generations of lymphocytes. In transplanted cells the signal of CFDA SE can be traced in vivo for weeks. The feasibility of using cell-permeant fluorescent tracers to follow cell division of natural killer (NK) cells, B cells, T cells, thymocytes, lymphocytes, fibroblasts and hematopoietic cells has been demonstrated with CFDA SE. For instance, researchers have used CFDA SE labeling to show that transplantable hematopoietic cells proliferate in vitro in response to stimulation by a growth factor cocktail. These studies helped provide direct evidence that the hematopoietic potential of cultured stem cells is limited by homing activity and not by proliferative capacity. Because the first division results in the largest change in fluorescence intensity, this method is particularly useful for detecting subsets of cells within a population that are resistant to cell division. The method is not limited to mammalian cells; it has also been applied to determine the number of cell divisions in stained Lactobacillus plantarum.

Figure 15.4.5 Tracking of asynchronous cell division using 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA SE or CFSE; C1157; V12883) labeling and flow cytometry. Cell division results in sequential halving of the initial fluorescence, resulting in a cellular fluorescence histogram. The peaks labeled 0, 1, 2, 3, 4 and 5 represent successive generations.

Figure 15.4.6 Following cell proliferation in human peripheral blood lymphocytes using the CellTrace CFSE Cell Proliferation Kit C34554). Human peripheral blood lymphocytes were harvested and stained with CellTrace CFSE (carboxyfluorescein diacetate, succinimidyl ester; 5(6)-CFDA, SE) on Day 0. A portion of the population was arrested at the parent generation using mitomycin C (red peak). The remainder of the sample was stimulated with phytohemagglutinin and allowed to proliferate for 5 days. Solid green peaks represent successive generations.

Like CFDA SE, the succinimidyl ester of Oregon Green 488 carboxylic acid diacetate (carboxy-DFFDA SE) should be a useful tool for following proliferating cells. This Oregon Green 488 probe passively diffuses into cells, where it is colorless and nonfluorescent until its acetate groups are removed by intracellular esterases to yield a highly green-fluorescent, amine-reactive dye. Upon reaction with intracellular amines, the probe forms Oregon Green 488 conjugates that are well-retained by cells. Unlike fluorescein derivatives, however, Oregon Green 488 derivatives exhibit bright green fluorescence that is not pH dependent at typical cellular pH values. Moreover, Oregon Green 488 probes are usually brighter and more photostable than fluorescein probes. We offer carboxy-DFFDA SE in a 1 mg unit size (O34550) and specially packaged in a set of 20 vials, each containing 50 µg (CellTrace Oregon Green 488 carboxylic acid diacetate succinimidyl ester, C34555).

The intracellular conjugates of 5-(and 6-)carboxyeosin diacetate succinimidyl ester (C22803) have absorption and emission spectra at longer wavelengths than CFDA SE, which may make this probe useful in combination with CFDA SE for studies of proliferation of mixed-cell populations. Eosin conjugates are more effective singlet-oxygen generators than are simple fluorescein derivatives, potentially resulting in their utility for photoablation of cells.

The succinimidyl ester of SNARF-1 carboxylic acid, acetate (S22801) is also designed to serve as a cell tracer and indicator of cell division. However, unlike the green-fluorescent CFDA SE–labeled cells, cells labeled with the succinimidyl ester of SNARF-1 carboxylic acid, acetate exhibit red fluorescence when excited near 488 nm. Although the fluorescence intensity of this SNARF derivative in cells may be weaker than that of cells labeled with CFDA SE, its red fluorescence is easily distinguished from the green fluorescence of CFDA SE–labeled cells. The SNARF dyes have been predominantly used as indicators of intracellular pH (pH Indicators—Chapter 20).

CellTrace Violet Cell Proliferation Kit

CellTrace Violet stain is an esterase-activated phenolic fluorophore with a succinimidyl ester substituent for coupling to cell surface and intracellular amines. It is functionally analogous to CFSE, equally partitioning between daughter cells during division resulting in successive two-fold reductions in cell-associated fluorescence intensity. When analyzed by flow cytometry, this progressive label partitioning provides a direct indication of cell proliferation status (Figure 15.4.7). In contrast to CFSE, CellTrace Violet stain is optimally excited by 405 nm violet diode lasers and generates blue fluorescence (emission peak ~455 nm). Consequently, it can be used in combination with CFSE to track cells from different origins after mixing or to analyze proliferation of GFP-expressing cells. The CellTrace Violet Cell Proliferation Kit (C34557) includes the CellTrace Violet stain together with dimethylsulfoxide (DMSO) for preparation of a stock solution.

Figure 15.4.7 Human peripheral blood lymphocytes were harvested and stained with CellTrace Violet stain. The violet peaks represent successive generations of cells stimulated with mouse anti–human CD3 and Interleukin-2 and grown in culture for 7 days. The peak outlined in black represents cells that were grown in culture for 7 days with no stimulus.

CyQUANT Cell Proliferation Assay Kit

Because cellular DNA content is highly regulated, it is closely proportional to cell number. Therefore, changes in nucleic acid content can serve as a sensitive indicator of overall cell proliferation, as well as of cytotoxic events or pathological abnormalities that affect cell proliferation. Our CyQUANT Cell Proliferation Assay Kit (C7026) provides an excellent method both for enumerating cells in a population and for measuring their proliferative activity. This assay is an important development for the rapid and quantitative screening of agents that affect cell proliferation. The CyQUANT assay is based on the use of our green-fluorescent CyQUANT GR dye, which exhibits strong fluorescence enhancement when bound to cellular nucleic acids. The assay protocol is simple: the culture medium is removed (nonadherent cells require brief centrifugation); the cells are frozen, thawed and lysed by addition of the CyQUANT cell buffer containing detergent and the CyQUANT GR dye; and fluorescence is then measured directly in a fluorometer or fluorescence microplate reader (Figure 15.4.8). No washing steps, growth medium changes or long incubations are required. The CyQUANT cell proliferation assay has a number of significant advantages over other proliferation assays:

• Sensitivity and linearity. The CyQUANT assay is linear from 50 or fewer cells to at least 50,000 cells in 200 µL volumes (Figure 15.4.9); increasing the dye concentration extends the linear range to at least 250,000 cells. Methods that employ Hoechst 33258 (H1398, H3569, H21491) or Hoechst 33342 (H1399; H3570, H21492) to measure cell number and proliferation are much less sensitive—detection limits of 500 cells for Hoechst 33258 or 2500 cells for Hoechst 33342 —and have much smaller effective ranges.
• No radioactivity. Unlike assays that measure ³H-thymidine incorporation, the CyQUANT assay does not require radioisotopes and thus does not have the hazards or the expense associated with use, storage and disposal of radioisotopes.
• Quick and easy protocol. The CyQUANT assay is a single-step procedure that requires no lengthy incubation steps and can be completed within an hour (Figure 15.4.8).
• Specificity and reliability. The assay is specific for total nucleic acids, with essentially no interference from other cell components. No wash steps are required because cellular growth media do not significantly interfere with CyQUANT GR fluorescence. The CyQUANT assay is reliable for cell quantitation, even without treatment to eliminate cellular RNA. However, addition of RNase or DNase permits the easy quantitation of DNA or RNA, respectively, in the sample.
• Convenience. Unlike assays that use tetrazolium salts, ³H-thymidine, BrdU, neutral red or methylene blue, the CyQUANT procedure is not dependent on cellular metabolism. Thus, cells can be frozen and stored prior to assaying, with no reduction in signal, or they can be assayed immediately after collection. Time-course assays are simplified because data obtained from stored samples taken at widely different time intervals can be assayed together with a single standard curve determination.
  

Figure 15.4.8 The simple procedure for using the CyQUANT Cell Proliferation Assay Kit (C7026).

Figure 15.4.9 Quantitation of NIH 3T3 fibroblasts using the CyQUANT Cell Proliferation Assay Kit (C7026). Fluorescence measurements were made using a microplate reader with excitation at 485 nm and emission detection at 530 nm. The linear range of the assay under these conditions is from 50 to 50,000 cells per 200 µL sample. The inset shows the linearity that can be obtained at very low numbers of cells.

We have found the CyQUANT Cell Proliferation Assay Kit to be useful for assaying widely disparate cell types, including:

• Human neonatal fibroblasts, keratinocytes, melanocytes, umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (DMVEC)
• Murine fibroblasts (NIH 3T3 and CRE BAG 2 cells) and myeloma (P3X63A68) cells
• Madin–Darby canine kidney (MDCK) cells
• Chinook salmon embryo (CHSE) cells
• Rat basophilic leukemia (RBL) and glioma (C6) cells

Determination of total cell number using the CyQUANT GR reagent is potentially useful for quantitating cell adhesion (see "Cell Adhesion" in Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6) and for determining the total number of cells in a tissue. Each CyQUANT Cell Proliferation Assay Kit (C7026) includes:

• CyQUANT GR reagent
• Cell-lysis buffer
• DNA standard for calibration
• Detailed protocols (CyQUANT Cell Proliferation Assay Kit)

The kit supplies sufficient materials for performing 1000 assays based on a 200 µL sample volume or a proportionately lower number of assays with a larger sample volume. The CyQUANT cell-lysis buffer (a 20X concentrate, C7027) is also available separately and has been formulated to produce efficient lysis, to protect nucleic acids from nuclease activity and to dissociate proteins that may interfere with dye binding to nucleic acids. It may prove generally useful in the development of other assays that require cell lysis.

CyQUANT NF Cell Proliferation Assay Kit

The CyQUANT NF Cell Proliferation Assay Kit provides a fast and sensitive method for counting cells in a population and measuring proliferation in microplate format. This assay can be completed in one hour, with no washes, cell lysis, long incubations or radioactivity required, and it is not dependent on physiological activities that may exhibit cell number–independent variability. The CyQUANT NF assay eliminates the freeze-thaw cell lysis step of the original CyQUANT cell proliferation assay by using a cell-permeant DNA-binding dye in combination with a plasma membrane–permeabilization reagent. The CyQUANT NF assay protocol requires only aspiration of growth medium (for adherent cells), replacement with dye binding solution, incubation for 30–60 minutes and then measurement of fluorescence in a microplate reader. The CyQUANT NF assay has a linear detection range from at least 100 to 20,000 cells per well in most cell lines using a 96-well microplate format and a 100 µL assay volume.

The CyQUANT NF Cell Proliferation Assay Kit can be used with either a 96-well or 384-well microplate format and is available in two configurations: a 200-assay kit (C35007) and a 1000-assay kit (C35006) for high-throughput applications. Each kit contains:

• CyQUANT NF dye reagent
• Dye delivery reagent
• Concentrated Hank's balanced salt solution (HBSS)
• Detailed protocols (CyQUANT NF Cell Proliferation Assay Kit)

CyQUANT Direct Cell Proliferation Assay Kit

CyQUANT Direct Cell Proliferation Assay is a fluorescence-based proliferation and cytotoxicity assay for microplate readers. The no-wash, homogenous format and fast add-mix-read protocol makes the CyQUANT Direct assay ideal for high-throughput screening (HTS) applications. The assay can be completed in one hour, with no washes, cell lysis, temperature equilibrations or radioactivity required, and the signal is stable for several hours to provide work-flow convenience. With a dynamic range from less than 50 to more than 20,000 cells of most adherent and suspension cell types, the CyQUANT Direct assay can be used in 96-, 384- or 1,536‑well microplate formats, and is compatible with most HTS and high-content screening (HCS) readers. Because the experimental protocol does not include a lysis step, the assay can conveniently be multiplexed using a spectrally distinct fluorescent or a luminescent readout.

The CyQUANT Direct Cell Proliferation Assay Kit is available in two configurations: a 10-plate assay kit (C35011) and a 100-plate assay kit (C35012) for high-throughput applications. Each kit contains:

• CyQUANT Direct nucleic acid stain
• CyQUANT Direct background suppressor
• Detailed protocols (CyQUANT Direct Cell Proliferation Assay Kit)

FluoReporter Blue Fluorometric Nucleic Acid Assay Kit

The FluoReporter Blue Fluorometric dsDNA Quantitation Kit (F2962) provides the protocols developed by Rago and colleagues for analyzing cellular DNA with the blue-fluorescent Hoechst 33258 nucleic acid stain. The kit enables researchers to detect ~10 ng of isolated calf thymus DNA or ~1000 mouse NIH 3T3 cells in a 200 µL sample (substantially lower levels are detectable using our CyQUANT Cell Proliferation Assay Kit described above).

With this kit, quantitation of cellular DNA is rapid, and all manipulations can be carried out in microplate wells. The cells are lysed by freezing them in distilled water, which circumvents the requirement for extraction procedures used in other Hoechst 33258 dye–based protocols. The diluted dye solution is then added to the lysed cells, and fluorescence is measured. Kit components include:

• Hoechst 33258 in DMSO/H₂O
• TNE buffer
• Detailed protocol (FluoReporter Blue Fluorometric dsDNA Quantitation Kit)

Each kit provides sufficient reagents for assaying approximately 2000 samples using a fluorescence microplate reader.

Vybrant MTT Cell Proliferation Assay Kit

Molecular Probes' convenient Vybrant MTT Cell Proliferation Assay Kit (V13154) simplifies the task of counting cells with a microplate absorbance reader. The colorimetric MTT assay, developed by Mosmann, is based on the conversion of the water-soluble MTT to an insoluble purple formazan. This formazan is then solubilized, and its concentration determined by optical density at 570 nm. The Vybrant MTT Cell Proliferation Assay Kit provides a sensitive assay with excellent linearity up to approximately 10⁶ cells per well. Each kit includes:

• MTT
• Sodium dodecyl sulfate (SDS)
• Detailed protocol (Vybrant MTT Cell Proliferation Assay Kit)

This kit provides sufficient materials for ~1000 assays using standard 96-well microplates. Numerous variations and modifications of the MTT assay have been published. In addition to dehydrogenases, MTT is reduced by glutathione S-transferase (GST). Therefore, MTT may not always be a reliable cell viability probe in cells treated with compounds that affect GST activity.

Vybrant DiI Cell-Labeling Solution

Analysis by mass spectrometry and HPLC indicates that the dye we use in our Vybrant DiI cell-labeling solution (V22885) is identical to the dye called PKH 26. DiI (PKH 26) is a red-fluorescent lipophilic tracer that, in addition to being extensively used for cell tracing (Tracers for Membrane Labeling—Section 14.4), has been utilized for generational analysis of cells undergoing division. Unlike the PKH 26 dye, which requires a special cell-labeling medium and low ionic strength for successful cell loading, our Vybrant DiI cell-labeling solution is simply added to cells in normal growth medium. Dividing cells distribute the lipophilic tracer approximately equally between daughter cells. It is usually possible to follow at least three or four generations of cells by flow cytometry, although asynchronous division times can quickly complicate the measurements. The dyes in our Vybrant DiO, Vybrant DiD and Vybrant CM-DiI Labeling solutions (V22886, V22887, V22888) may have similar utility for tracing cells through cell division. CM-DiI contains a thiol-reactive chloromethyl that allows the dye to covalently bind to cellular thiols. Thus, unlike other membrane stains, this label is well retained in some cells throughout fixation and permeabilization steps; see Tracers for Membrane Labeling—Section 14.4 for more information.

CountBright Absolute Counting Beads

Flow cytometry provides a rapid method for quantitating cell characteristics, however most flow cytometers cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have been widely used in quantitating cell populations and disease progression and are generally obtained either by combining a separate cell concentration determination from a hematology analyzer with flow cytometry population data (multiple-platform testing) or by adding an internal microsphere counting standard to the flow cytometry sample (single-platform testing). The single-platform method is preferred as it is technically less complicated and more accurate than multiple-platform testing.

CountBright absolute counting beads (C36950) are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths and contain a known concentration of microspheres. For absolute counts, a specific volume of the microsphere suspension is added to a specific volume of sample, such that the ratio of sample volume to microsphere volume is known. The volume of sample analyzed can be calculated from the number of microsphere events and then used with cell events to determine cell concentration. In general, at least 1000 bead events should be acquired to assure a statistically significant determination of sample volume. Sufficient reagents are provided for 100 flow cytometry assays, each using 50 µL of counting beads per test.

CountBright absolute counting beads are broadly fluorescent and can be used with either a fluorescence or scatter threshold. Fluorescence can be excited by wavelengths from UV to 635 nm; fluorescence emission can be read between 385 nm and 800 nm. The fluorescence intensity of the microspheres has been adjusted to be about 5–50 times brighter than the anticipated intensities of typically stained cells. When using a scatter threshold, the microsphere signal should be above the threshold. The microspheres can be gated by a single parameter, but a combination of parameters can be used to resolve microspheres from cells and other events.

CountBright absolute counting beads can be used with any sample type, including no-wash/lysed whole blood. The microspheres in the reagents are approximately 7 µm in diameter and have settling properties similar to lymphocytes. The accuracy of cell counts based on CountBright absolute counting beads depends on sample handling and the precise delivery of the volume of beads. The CountBright absolute counting beads must be mixed well to assure a uniform suspension of microspheres. After vortexing for 30 seconds, the microsphere suspension can be pipetted by standard techniques; however, more viscous solutions such as blood require reverse pipetting for accurate volume delivery. Cell suspensions may be diluted but should be assayed without wash steps. Other sample preparation steps that can lead to cell or microsphere loss should also be avoided. For antibody protocols, CountBright absolute counting beads should be used with reagents titered for no-wash staining.

Detecting Bacteria, Yeast and Plankton Using Nucleic Acid Stains

We recommend our SYTO nucleic acid stains (see the complete list in Cell-permeant cyanine nucleic acid stains—Table 8.3) for simple detection of the presence of bacteria, yeast and other microbial cells (, , ). The SYTO dyes are essentially nonfluorescent except when bound to nucleic acids, where they become highly fluorescent, often with quantum yields exceeding 0.5. Consequently, it is usually not necessary to remove unbound stains before analysis. SYTO dyes are available with blue, green, orange or red fluorescence. The SYTO dyes rapidly penetrate the membranes of almost all cells, including bacteria and yeast. The various cell types can often be identified by their characteristic morphology or, in the case of flow cytometric applications, by their light-scattering properties.

The SYTO 11 and SYTO 13 green-fluorescent nucleic acid stains show exceptional ability to penetrate tissues for at least 100 µm, including untreated, unfixed human brain tissue, where they were used to enumerate cells by confocal laser-scanning microscopy. Simultaneous labeling with a green-fluorescent SYTO dye and a red-fluorescent nucleic acid stain—most often propidium iodide, ethidium homodimer-1 or -2, TOTO-3 or TO-PRO-3 (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2, Properties of classic nucleic acid stains—Table 8.4)—is frequently used to assess cell viability (Viability and Cytotoxicity Assay Reagents—Section 15.2). Although some of the SYTO dyes show higher quantum yields on DNA or RNA, they should not be considered specific stains for either of these nucleic acids. We offer four different SYTO Nucleic Acid Stain Sampler Kits (S11350, S7572, S11360, S11340; Nucleic Acid Stains—Section 8.1).

In addition to its use for cell-cycle analysis (see below) and nuclear staining, DAPI (D1306, D3571, D21490) is frequently employed for DNA content–based counting of bacterial cells and for detecting malarial infections by fluorescence microscopy.

BacLight Bacterial Stains

The BacLight Green and BacLight Red bacterial stains (B35000, B35001) are fluorescent, non-nucleic acid labeling reagents for detecting and monitoring bacteria. Bacteria stained with the BacLight Green and BacLight Red bacterial stains exhibit bright green (excitation/emission maxima ~480/516 nm) and red (excitation/emission maxima ~480/516 nm) fluorescence, respectively, and can be resolved simultaneously using the appropriate flow cytometry channels. Although these dyes were specifically chosen for flow cytometry applications, bacteria stained with these BacLight reagents can also be visualized by fluorescence microscopy with only minor, if any, adjustments in the staining concentrations. Furthermore, the BacLight bacterial staining patterns are compatible with formaldehyde or alcohol fixation methods.

These BacLight bacterial stains efficiently label a variety of different bacteria species. The intensity of the staining appears to depend on several factors, including gram character, outer membrane composition and overall membrane integrity. In the species we tested, gram-positive bacteria generally exhibited brighter fluorescence than gram-negative bacteria, and cells with compromised membranes accumulated more dye than intact cells (Figure 15.4.10).

Figure 15.4.10 Flow cytometry histograms showing fluorescence of live and dead gram-positive and gram-negative bacteria stained with the BacLight bacterial stains. Untreated and alcohol-treated gram-positive (Staphylococcus aureus, (A and C)) and gram-negative (Escherichia coli, (B and D)) bacteria were each stained separately with 100 nM of either the BacLight Green (A and B) or the BacLight Red (C and D) bacterial stains (B35000, B35001) in 0.85% NaCl buffer and then analyzed by flow cytometry. The histograms for the untreated (colored histogram curve) and alcohol-treated (uncolored histogram curve) bacteria samples were overlaid for each species and BacLight bacterial stain.

Bacteria Counting Kit

Accurate enumeration of low numbers of bacteria in samples must be performed daily in many quality-control laboratories. To facilitate this determination by flow cytometry (Figure 15.4.11), we have developed the Bacteria Counting Kit (B7277), which provides:

• Cell-permeant, green-fluorescent SYTO BC nucleic acid stain to label bacteria
• Fluorescent polystyrene microspheres to calibrate the volume of bacterial suspension analyzed
• Detailed protocols (Bacteria Counting Kit) 

SYTO BC dye, which is also available separately (S34855, Nucleic Acid Stains—Section 8.1), is a high-affinity nucleic acid stain that easily penetrates both gram-negative and gram-positive bacteria, producing an exceptionally bright green-fluorescent signal. The calibrated suspension of polystyrene microspheres contains beads that exhibit a uniform density, low-level fluorescence and optimal size to clearly separate the light scattering of the microspheres from that of most bacteria.

The Bacteria Counting Kit is particularly valuable for monitoring antibiotic sensitivity because it provides a convenient and accurate means for assessing changes in a bacterial population over time. A sample of the population is simply diluted, stained briefly with the SYTO BC dye, mixed with a fixed number of microspheres and analyzed on a flow cytometer. Signals from both the stained bacteria and the beads are easily detected in the green–fluorescence channel of most flow cytometers and can be distinguished on a plot of forward scatter versus fluorescence (Figure 15.4.12). The density of the bacteria in the sample can be determined from the ratio of bacterial signals to microsphere signals in the cytogram. The Bacteria Counting Kit can be used with a variety of gram-negative and gram-positive species of bacteria and provides sufficient reagents for approximately 100 flow cytometry assays.

The fluorescent microspheres in our Bacteria Counting Kit have also been recommended for the enumeration of yeast. We offer a wide selection of labeled beads (Microspheres—Section 6.5, Fluorescence Microscopy Reference Standards and Antifade Reagents—Section 23.1, Flow Cytometry Reference Standards—Section 23.2) that may also prove useful for yeast quantitation and viability assays by flow cytometry.

Figure 15.4.11 Detection and counting of bacteria in milk using the Bacteria Counting Kit (B7277). Equal numbers of bacteria were suspended in 150 mM NaCl or a mixture of milk and 150 mM NaCl and assayed using the protocol provided with the kit. As shown in the lower bar chart, the presence of milk does not affect the outcome of the assay. The upper panels plot green fluorescence versus side scatter for: A) bacteria alone, B) bacteria alone, C) milk alone and D) bacteria mixed with milk (spiked milk). Panels B–D also contain reference beads, which appear in the upper right corner of the respective plots.

Figure 15.4.12 Flow cytometric enumeration of Bacillus cereus using the Bacteria Counting Kit (B7277). In this plot of forward scatter versus fluorescence, signals in the upper lefthand frame represent bacteria stained with SYTO BC bacteria stain; signals in the lower righthand frame represent microsphere particles, which serve as a standard used to indicate sample volume.

Cell Culture Contamination Detection Kit

Molecular Probes Cell Culture Contamination Detection Kit (C7028) uses a simple and effective procedure for visually screening cell cultures for contamination by yeast (and other fungi) or by gram-negative or gram-positive bacteria. This kit not only serves to detect the contaminants, but also identifies the contaminant type, enabling the researcher to choose an appropriate course of action.

A sample of the suspected culture is subjected to two slide-staining protocols. One sample slide is stained with Calcofluor White M2R, a UV light–excitable, blue-fluorescent stain specific for fungal cell walls. A second slide is stained with SYTO 9 nucleic acid stain to identify all bacteria irrespective of gram signature, and also with the Texas Red-X conjugate of wheat germ agglutinin (WGA), which selectively stains gram-positive bacteria. Gram-positive bacteria on the second slide typically exhibit a green-fluorescent interior with red-fluorescent cell-surface staining, whereas gram-negative bacteria show only green-fluorescent interior staining. Staining and examination of the slides under a fluorescence microscope can be performed in less than one hour. Each Cell Culture Contamination Detection Kit contains:

• Green-fluorescent SYTO 9 nucleic acid stain
• Blue-fluorescent Calcofluor White M2R fungal cell wall stain
• Red-fluorescent Texas Red-X WGA, for positive identification of gram-positive bacteria
• Buffer for reconstituting Texas Red-X WGA
• Detailed protocols (Cell Culture Contamination Detection Kit)

This kit provides sufficient material for approximately 200 contamination detection assays. The SYTO 9 nucleic acid stain, which is also available separately (S34854, Viability and Cytotoxicity Assay Reagents—Section 15.2), has been used to detect lactic acid–producing bacteria in wine.

MycoFluor Mycoplasma Detection Kit

Mycoplasma infections are generally difficult or impossible to detect during routine work with cultured cells because these intracellular pathogens cannot be observed by standard light microscopy. It is not surprising then that mycoplasma infections are relatively common. Estimates of contaminated cultures in the United States range from 5% to 35%, whereas the contamination rate is postulated to be much higher in those countries where systematic detection and elimination are not practiced. Not only do mycoplasma cause physiological and morphological distortions that can affect experimental results, but contamination can quickly spread to other cell lines. Mycoplasma infections can be detected with Hoechst dyes () or with DAPI (Nucleic Acid Stains—Section 8.1). Hoechst 33258, either alone or in combination with merocyanine 540 (M24571, Generating and Detecting Reactive Oxygen Species—Section 18.2), has been utilized to eradicate mycoplasma infections from cell cultures.

The MycoFluor Mycoplasma Detection Kit (M7006) provides an extremely rapid and sensitive fluorescence microscopy–based assay for the visual identification of mycoplasma infection in laboratory cell cultures and media. In order to detect mycoplasma, the fluorescent MycoFluor reagent is added directly to the culture medium, with or without cells present, and the stained sample is then examined under a fluorescence microscope. The test for the presence of mycoplasma in live or fixed cell cultures takes about 15 minutes from when the reagent is added until when the sample is viewed with a fluorescence microscope equipped with DAPI optical filters. The detection of mycoplasma in cell media requires about 30 minutes, depending on the amount of centrifugation required to concentrate potential contaminants.

Also provided with this kit are mycoplasma MORFS (Microscopic Optical Replicas for Fluorescence assays), which serve as inert positive controls that mimic the size, shape and fluorescence intensity of mycoplasma stained with the blue-fluorescent MycoFluor reagent and viewed by fluorescence microscopy. The optical properties of the mycoplasma MORFS enable the researcher to discriminate between stained mycoplasma and other fluorescent material without introducing infectious biological agents into the laboratory environment. No previous experience with mycoplasma testing is required.

Each MycoFluor Mycoplasma Detection Kit supplies sufficient materials for at least 100 tests of live cells, fixed cells or culture media. Kit contents include:

• Concentrated MycoFluor reagent
• Suspension of mycoplasma MORFS
• Coverslip sealant
• Cotton swab
• Reference micrographs
• Detailed protocols (MycoFluor Mycoplasma Detection Kit) 

Alternatively, the LIVE/DEAD BacLight Bacterial Viability Kits (L7007, L7012, L13152; Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) are useful for detecting mycoplasma infections. Researchers have determined that the reagents in our LIVE/DEAD BacLight Kits can be used for viability determinations in Eurioplasma eurilytica and Mycoplasma hominus mycoplasma and in the cysts of the protozoan parasite Giardia muris ().

Detecting Viruses Using Nucleic Acid Stains

Because of their small size, direct detection of viruses by fluorescence requires highly sensitive reagents or, much more commonly, an amplification scheme. However, direct enumeration of marine viral abundance in seawater using SYBR Green I nucleic acid gel stain (S7563, S7567, S7585; Nucleic Acid Quantitation in Solution—Section 8.3; ), YO-PRO-1 (Y3603, Nucleic Acid Stains—Section 8.1) and DAPI (D1306, D3571, D21490) has been reported.

The slow off-rate of our dimeric nucleic acid stains, such as TOTO-1 and YOYO-1 (T3600, Y3601), has permitted their use for labeling nucleic acids, including viral RNA, prior to microinjection into live cells to follow their trafficking. Similar staining techniques may permit tracing of viral uptake and transport by live cells.

The Countess automated cell counter (C10227, C10310, C10311; Figure 15.4.13) uses Trypan Blue staining combined with a sophisticated image analysis algorithm to count all cells in a sample―even mildly clumpy samples―as well as assess viability and measure average cell size. The Countess instrument produces accurate cell and viability counts and completes all calculations in 30 seconds, using as little as 5 µL of your sample; a cell viability percentage value is calculated automatically.

The measurement range of the Countess automated cell counter extends from 1 × 10⁴ to 1 × 10⁷ cells/mL, with an optimal range from 1 × 10⁵ to 4 × 10⁶ cells/mL, which is broader than that of a hemocytometer. Furthermore, the Countess instrument counts cell size from 5 to 60 µm and counts beads from 4.5 to 60 µm. Countess Test Beads (C10284) provide a reliable and rapid calibration standard for users who choose to operate the instrument outside its default parameters. The Countess instrument has been validated for use with more than 20 common cell types, including primary cells, blood cells, yeast cells (without viability assessment), insect cells and fish cells.

The Countess automated cell counter does not require a computer. Disposable chamber slides (C10228, C10312, C10313, C10314, C10315) provide rapid setup, with no cleaning, buffers or maintenance required other than battery replacement after approximately 5 years of use. Trypan Blue stain is also available as a stand-alone reagent (T10282).

Figure 15.4.13 The Countess automated cell counter provides fast, easy and accurate cell counting without using a hemocytometer.

In 2008, Miyawaki and colleagues developed the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI), a fluorescent protein (FP)–based sensor that employs a Red Fluorescent Protein (RFP) and a Green Fluorescent Protein (GFP), each of which is fused to one of two different regulators of the cell cycle: Cdt1 and geminin. Ubiquitin E3 ligases add ubiquitin to the Cdt1–GFP and geminin–RFP fusions, thereby targeting these proteins for proteasomal degradation. Temporal regulation of E3 ligase activity results in the biphasic cycling of the geminin and Cdt1 fusions through the cell cycle.

In the G₁ phase, the geminin–GFP fusion is degraded, leaving only the Cdt1–RFP fusion and resulting in red-fluorescent nuclei. During the G₁/S transition, Cdt1 levels decrease and geminin levels increase. Because both proteins are present in the cells, both GFP and RFP fluorescence is visible and the cell nuclei appear yellow when the green and red images are overlaid. In the S, G₂, and M phases, the Cdt1–RFP fusion is degraded, leaving only the geminin–GFP fusion and resulting in green-fluorescent nuclei. This dynamic color change from red to yellow to green serves as an indicator of the progression through cell cycle and division (Figure 15.4.14).

The Premo FUCCI cell cycle sensor (P36237, P36238) combines the Cdt1 and geminin FP constructs with the powerful BacMam gene delivery and expression system (BacMam Gene Delivery and Expression Technology—Note 11.1). The genetically encoded and pre-packaged reagents are ready for immediate use, eliminating the need to purify plasmid. The BacMam reagent is simply added to cells for 1–2 hours, after which the cells are treated with an enhancer reagent for another 1–2 hours, washed, incubated overnight and then visualized using fluorescence microscopy or high-throughput imaging platforms (Figure 15.4.14). BacMam transduction is efficient and reproducible in most cell types, including primary and stem cells, without apparent cytotoxic effects.

Premo FUCCI cell cycle sensor is designed for live-cell imaging of cell cycle progression and can be used to assess the effect of drugs, siRNA or other factors on the transition of cells through the cell cycle. The fluorescence signals from geminin-GFP and Cdt1-RFP (Figure 15.4.15) have been demonstrated to be resistant to fixation with 4% formaldehyde and permeabilization with 0.1% Triton X-100, thereby enabling processing of labeled cells with antibodies to other cellular targets. Each kit contains all of the components needed to label cells with the Premo FUCCI cell cycle sensor using a transduction volume of 2 mL; however, the protocol can easily be adjusted for larger or smaller volumes.

Figure 15.4.14. Imaging cell-cycle progression in live cells with Premo FUCCI Cell Cycle Sensor. A) Schematic of cell-cycle progression with nuclear fluorescence changes. B) U2OS cells were transduced with Premo FUCCI Cell Cycle Sensor, and images were collected over 15 hours.

Figure 15.4.15 Fluorescence excitation and emission spectra for A) Premo geminin–GFP and B) Premo Cdt1–RFP.

Live-cell studies of cellular DNA content and cell-cycle distribution are useful for investigating tumor behavior and suppressor gene mechanisms, for monitoring apoptosis and for detecting variations in growth patterns arising from a variety of physical, chemical and biological means. In a given population, cells will be distributed among three major phases of the cell cycle: G₀/G₁ phase (one set of paired chromosomes per cell), S phase (DNA synthesis with variable amount of DNA) and G₂/M phase (two sets of paired chromosomes per cell, prior to cell division). DNA content can be measured using fluorescent, DNA-selective stains that exhibit emission signals proportional to DNA mass. Flow cytometric analysis of these stained populations is then used to produce a frequency histogram that reveals the various phases of the cell cycle. This analysis is typically performed on permeabilized or fixed cells using a cell-impermeant nucleic acid stain, but is also possible using live cells and a cell-permeant nucleic acid stain. While the choices for fixed cell staining are varied, there are only a few examples of useful cell-permeant nucleic acid stains.

Vybrant DyeCycle Stains

All Vybrant DyeCycle stains are DNA-selective, cell membrane–permeant and nonfluorescent until bound to double-stranded DNA. Once bound to DNA, these dyes emit a fluorescence signal that is proportional to DNA mass. Staining cells with the Vybrant DyeCycle stains is simple: suspended cells are incubated in the presence of a Vybrant DyeCycle stain, and fluorescence is measured directly—no additional treatment or centrifugation is required. Fluorescence data can then be used to generate a frequency histogram that reveals the various phases of the cell cycle.

We offer four Vybrant DyeCycle stains for flow cytometry analysis of DNA content in live cells:

• Vybrant DyeCycle Violet stain (V35003)
• Vybrant DyeCycle Green stain (V35004)
• Vybrant DyeCycle Orange stain (V35005)
• Vybrant DyeCycle Ruby stain (V10273, V10309)

The Vybrant DyeCycle stains spectrally match the commonly available excitation sources, placing cell-cycle studies within reach of all flow cytometrists and allowing simultaneous staining of the cell population for other parameters. Vybrant DyeCycle Violet stain (excitation/emission maxima ~396/437 nm) is well suited for the 405 nm laser line but can also be used with UV excitation. Vybrant DyeCycle Green stain (excitation/emission maxima ~506/534 nm) is efficiently excited with the 488 nm spectral line of the argon-ion laser. Vybrant DyeCycle Orange stain (excitation/emission maxima ~519/563 nm) can be excited using either the 488 nm or 532 nm laser lines. Vybrant DyeCycle Ruby stain (excitation/emission maxima ~638/686 nm) can be excited using the 635 nm red diode laser.

FxCycle Stains for Cell-Cycle Analysis

FxCycle Violet stain (F10347) is a violet diode–excited dye used for cell-cycle analysis in fixed cells. FxCycle Violet stain (4',6-diamidino-2-phenylindole, dihydrochloride or DAPI) preferentially stains dsDNA, exhibiting a ~20-fold fluorescence enhancement upon binding. Using FxCycle Violet for cell cycle analysis increases the ability to multiplex by freeing up the 488 nm and 633 nm lasers for other cellular analyses, such as immunophenotyping, apoptosis analysis and dead cell discrimination. FxCycle Violet Stain overlaps less with other fluorescence detection channels than other commonly used dyes (propidium iodide and 7-AAD), resulting in minimal compensation requirements and more accurate data.

FxCycle Far Red stain (F10348) is useful for flow cytometric analysis of DNA content in fixed cells. Because FxCycle Far Red stain binds to both RNA as well as DNA, the addition of RNase A is required for DNA content analysis. This dye takes advantage of the commonly available 633/635 nm excitation sources, with emission around 660 nm, and is a good choice for DNA content analysis in multicolor cell-cycle studies.

SYTOX Green Nucleic Acid Stains

SYTOX Green nucleic acid stain (S7020) is particularly useful for cell-cycle analysis on RNase-treated fixed cells (Figure 15.4.16). In particular, the SYTOX Green dye produces lower coefficients of variation than propidium iodide (P1304MP, P3566, P21493; Nucleic Acid Stains—Section 8.1), leading to improved resolution of cell phase. Figure 15.4.16 shows a comparison of DNA content histograms obtained with SYTOX Green nucleic acid stain and propidium iodide after flow cytometric analysis.

Figure 15.4.16 Comparison of DNA content histograms obtained with A) SYTOX Green nucleic acid stain (S7020) and B) propidium iodide (P1304MP, P3566, P21493). Human B cells were suspended in permeabilizing buffer (100 mM Tris, pH 7.4, 154 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 0.1% Nonidet P-40) and then stained for 15 minutes with 0.5 µM SYTOX Green or 5 µM propidium iodide. Flow cytometric analysis of the stained cells was carried out with excitation at 488 nm. SYTOX Green staining produces a significantly narrower G₁ phase peak, indicated by the smaller coefficient of variation (CV).

Hoechst 33258, Hoechst 33342 and DAPI

The nucleic acid stains most frequently used for cell-cycle analysis—Hoechst 33258 (H1398, H3569, H21491), Hoechst 33342 (H1399, H3570, H21492) and DAPI (D1306, D3571, D21490)—bind to the minor groove of DNA at AT-rich sequences. Hoechst 33342, which more rapidly permeates cells than Hoechst 33258, is commonly used for determining the DNA content of viable cells without detergent treatment or fixation (Figure 15.4.17). The Hoechst dyes and DAPI can be excited with a mercury-arc lamp, the UV spectral lines of the argon-ion laser or the 325 nm spectral line of the He-Cd laser. These blue-fluorescent nucleic acid stains preferentially bind to AT-rich sequences and also exhibit higher quantum yields when bound to AT-rich nucleic acids, thus introducing a strong bias into the measurements of nuclear DNA content. As a consequence, data obtained with Hoechst 33342 and DAPI correlate very well with each other but less well with data obtained with propidium iodide, a red-fluorescent, cell-impermeant nucleic acid stain (P1304MP, P3566, P21493; Nucleic Acid Stains—Section 8.1). Hoechst 33342 is used in the high-speed sorting of X chromosome– and Y chromosome–bearing sperm based on their DNA content.

Figure 15.4.17 DNA content histogram for WIL2S cells. The cells were fixed in ethanol, treated with RNase and stained with 2 µg/mL Hoechst 33342 (H1399, H3570, H21492). Flow cytometric analysis of the stained cells was carried out using excitation at 350 nm.

Histones are core proteins of DNA in eukaryotic cells. This histones are organized as octamers, around which the DNA is wrapped. The phosphorylation of histone 3 (H3) is involved in condensation of chromatin during mitosis and reaches a peak during mitosis. Mitotic H3 phosphorylation occurs at Ser10 of the amino terminus, and there is a tight correlation between H3 (Ser10) phosphorylation, chromosome condensation and segregation during mitosis. This event can serve as an indication of mitotic progression or inhibition within the context of drug profiling.

The HCS Mitotic Index Kit (H10293) was developed to measure mitotic cells using automated imaging and analysis and can be combined with other measurements such as DNA profiling, general cytotoxicity or immunocytochemical detection of choice targets. This kit includes sufficient reagents for two 96-well plate assays, including:

• Polyclonal rabbit anti–phospho-H3 antibody
• Alexa Fluor 488 goat anti–rabbit IgG antibody
• DAPI
• HCS NuclearMask Deep Red stain
• Detailed protocols (HCS Mitotic Index Kit)

The primary antibody against phosphorylated histone H3 (Ser10) serves as a sensitive index of mitosis and is detected using the green-fluorescent Alexa Fluor 488 secondary antibody. The blue-fluorescent DAPI and near- infrared–fluorescent HCS NuclearMask Deep Red stain provide two choices for DNA profiling and cell demarcation during image analysis. The HCS Mitotic Index Kit represents a powerful image-based assay for the identification of compounds that affect mitotic progression (Figure 15.4.18).

Figure 15.4.18 Imaging of nocodazole-induced mitotic arrest in A549 cells using the HCS Mitotic Index Kit (H10293). A) Dose response to 0–1,000 nM nocodazole after 24 hr incubation. The curve generated by nonlinear regression was used to determine the EC₅₀ value for nocodazole. B) Quantitation of cells treated with 500 nM nocodazole for 24 hr. Nuclear segmentation and DNA content were measured using DAPI or HCS NuclearMask Deep Red stain. The strong increase in both phosphohistone 3 and DNA staining is indicative of mitotic cells.