Tracking Internalization in Live Cells With pHrodo Dyes

pHrodo Red and pHrodo Green pH Indicators

(See a complete list of products discussed in this article.)

The plasma membrane not only encloses the cytosol, but also serves as a formidable barrier to the extracellular environment. Endocytosis is the process by which eukaryotic cells can overcome this barrier and engulf material—including hydrated ions, small polar molecules, large biomolecules such as antibodies, and even other cells—that cannot freely cross the plasma membrane. Endocytosis is essential for normal cell functioning, and many viruses and bacteria have evolved mechanisms to hijack endocytic pathways in order to gain entry into the cell. The pH-sensitive pHrodo dyes, when conjugated to dextrans, antibodies, or microbes, can be used to specifically detect endocytosis and phagocytosis (engulfment of larger particles) in live cells. Supplementing our classic pHrodo Red indicator, the new pHrodo Green indicator offers increased flexibility for multicolor experiments.

Highly Specific Detection of Endocytosis

Molecules and pathogens that encounter the plasma membrane are funneled into membrane invaginations that subsequently bud inward to form endosomes (or phagosomes in the case of microbe internalization by macrophages). The endocytic pathway of mammalian cells is composed of three distinct membrane compartments, each with increasing acidity. Cargo is first internalized into mildly acidic early endosomes, where the molecules are either recycled back to the cell surface or delivered to late endosomes. Late endosomes (~pH 5.5) mediate the delivery of the material to acidic lysosomes (~pH 4.8) for degradation.

This acidification of endocytic compartments, a hallmark of endocytosis, is exploited by the pHrodo dyes, which are nonfluorescent at neutral pH and exhibit increasing fluorescence (red or green) as the pH becomes more acidic (Figure 1). Consequently, pHrodo dyes, when conjugated to dextrans, proteins, or other particles, can be used as highly specific sensors of endocytic and phagocytic internalization and lysosomal sequestration in live cells (Figure 2), offering a superior alternative to conjugates of other fluorescent dyes such as fluorescein and tetramethylrhodamine.

Because pHrodo fluorescence provides a positive indication of endocytosis, pHrodo indicators are desirable over pH-based negative indicators (in which fluorescence is quenched upon acidification), including fluorescein and Oregon Green dyes. The minimal pHrodo dye fluorescence at neutral pH also eliminates the need for wash steps and quencher dyes because any noninternalized dye will be essentially nonfluorescent.

Figure 1. pH sensitivity of pHrodo Red and pHrodo Green indicators.

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Figure 2. Tracking the internalization of biomolecules using pHrodo Green and pHrodo Red indicators.

Imaging Phagocytosis With Low Background

We have developed a no-wash, no-quench assay for phagocytosis using Escherichia coli, Staphylococcus aureus, or yeast zymosan conjugated to the pHrodo Red or Green indicators (Figure 3). This BioParticles product line consists of a series of fluorescently labeled killed bacteria and yeast (zymosan), offering a variety of sizes, shapes, and natural antigenicities. Because the microbes outside the cell exhibit minimal signal at neutral pH, there is no need to wash, quench, or even remove the extracellular particles. In addition to the pHrodo conjugates, BioParticles products are available with other fluorophores, including Alexa Fluor, fluorescein, BODIPY FL, tetramethylrhodamine, and Texas Red dyes.

Fluorescent bacteria and yeast particles have been employed in a variety of published studies using fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry (Figure 4). These include the detection of yeast phagocytosis by macrophages [1] and human neutrophils [2], the study of the effects of different opsonization procedures on the efficiency of phagocytosis of pathogenic bacteria [3] and yeast [2], the investigation of the kinetics of phagocytosis degranulation and actin polymerization in stimulated leukocytes [2], and the analysis of molecular defects in phagocytic function [4].

Figure 3. Live-cell endocytosis with pHrodo Green and pHrodo Red indicators. A431 cells were incubated with a 100 μg/mL solution of pHrodo Green or pHrodo Red 10,000 MW dextran  in Live Cell Imaging Solution (LCIS) for 30 min at 37°C (top row), a 1 mg/mL solution of pHrodo Green or pHrodo Red E. coli BioParticles conjugate in LCIS for 60 min at 37°C (middle row), or a 0.5 mg/mL solution of pHrodo Green or pHrodo Red zymosan BioParticles conjugate in LCIS for 60 min at 37°C (bottom row). After incubation, cells were stained with 2 drops of NucBlue Live Cell Stain per mL of cell sample and imaged by fluorescence microscopy using FITC or TRITC optical filter sets.

Figure 4. Flow cytometry analysis showing increased fluorescence of granulocytes treated with pHrodo Red BioParticles conjugates. A whole blood sample was collected and treated with heparin, and two 100 µL aliquots were prepared. Both aliquots were treated with pHrodo Red E. coli BioParticles and vortexed. One sample was placed in a 37°C water bath, and the other sample (negative control) was placed in an ice bath. After a 15 min incubation, red blood cells were lysed with an ammonium chloride–based lysis buffer. The samples were centrifuged for 5 min at 500 x g, washed once, and resuspended in HBSS. The samples were then analyzed on a BD FACSCalibur cytometer using a 488 nm argon laser and 564–606 nm emission filter. The sample incubated at 37°C shows the increased fluorescence of the phagocytosed pHrodo Red BioParticles (red), in contrast to the negative control sample, which was kept on ice to inhibit phagocytosis (blue).

Create Your Own pHrodo Bioconjugates

The amine-reactive ester and thiol-reactive maleimide forms of the pHrodo dye—now offered with green or red fluorescence—allow you to create your own bioconjugates, including pHrodo dye–labeled antibodies, peptides, or viruses. Our protein labeling kits allow you to easily conjugate the protein of your choice with pHrodo dyes. Because the pHrodo dye is extremely sensitive to its local environment, the pH response of each bioconjugate must be individually determined.

pHrodo dye conjugated to either avidin or the trastuzumab antibody—molecules that specifically bind to receptors overexpressed on tumor cells—enabled visualization of the cancer cells in tumor-bearing mice with high target-to-background ratios [5]. pHrodo Red succinimidyl ester has also been used to label dexamethasone-treated thymocytes for flow cytometric analysis of phagocytosis by macrophages [6], and for live-cell confocal imaging of antigen transfer from human B lymphocytes to macrophages [7].

Explore the Role of Endocytosis in Cell Health

pHrodo pH indicators (Table 1) are ideal tools for the study of endocytosis and its regulation in both normal cell development and disease processes. With the new pHrodo Green indicator and conjugates, you can now incorporate pHrodo dyes into multiplex experiments with red-fluorescent markers such as RFP, MitoTracker Red CMXRos, and CellROX Deep Red reagent, or with blue-fluorescent probes such as NucBlue live cell stain.

We offer an array of fluorescent live-cell probes for analyzing the internalization of biomolecules by cells; these include fluorescent ligands for studying receptor-mediated endocytosis, membrane markers of endocytosis, and CellLight targeted fluorescent protein conjugates for labeling cell organelles and cytoskeletal structures.

Table 1. Spectral characteristics and applications of pHrodo conjugates, reactive dyes, and labeling kit.

References

1. Ragsdale RL, Grasso RJ (1989) J Immunol Methods 123:259–267.
2. Oben JA, Foreman JC (1988) J Immunol Methods 112:99–103.
3. Sjursen H, Bjerknes R, Halstensen A et al. (1989) J Immunol Methods 116:235–243.
4. Deriy LV, Gomez EA, Zhang G et al. (2009) J Biol Chem 284:35926–35938.
5. Ogawa M, Kosaka N, Regino CA et al. (2010) Mol Biosyst 6:888–893.
6. Miksa M, Komura H, Wu R et al. (2009) J Immunol Methods 342:71–77.
7. Harvey BP, Quan TE, Rudenga BJ (2008) J Immunol 181: p. 4043–4051.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.