Cell Meter™ Fluorimetric Phagocytosis Assay Kit Red Fluorescence

Examination of phagocytosis in RAW 264.7 cells using Cell Meter™ Fluorimetric Phagocytosis Assay Kit (Cat# 21225). RAW 264.7 cells were incubated with (B) or without (A) Cytochalasin D for 30 min before Protonex™ 600 Red-Latex Beads in growth medium was added and incubated for 4 hours before Cell Tracker was added and incubated for 30 minutes. The images were taken using Keyence Fluorescence microscopy.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
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Spectral properties

Excitation (nm)	576
Emission (nm)	597

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Excitation (nm)

576

Emission (nm)

597

Phagocytosis is defined as the cellular uptake of particles within a plasma-membrane envelope by a cell. The process of phagocytosis is critical in the innate immune response by engulfment and destruction of invading microorganisms. Phagocytosis is also required in maintaining tissue homeostasis and remodeling by the clearance of apoptotic bodies. The uptake mechanism of phagocytosis depends on the size of the particles, receptor-ligand interactions, and involvement of the cytoskeleton. Once internalized, the phagosome fuses with lysosomes to form secondary phagolysosome for digestion, resulting in progressive decrease of pH. Our Cell Meter™ Fluorimetric Phagocytosis Assay Kit utilizes a unique pH dependent Protonex™ 600 Red-latex bead conjugates. The beads are in ready to use suspension. Unlike most of the existing fluorescent dyes, the Protonex™ 600 Red-latex bead conjugate is non-fluorescent outside of the cells. However, its fluorescence dramatically increases as they are inside the acidic phagosomes/phagolysosomes. This characteristic makes it easy to use without trypan blue quenching step and a robust tool to study phagocytosis and its regulations. Cell Meter™ Fluorimetric Phagocytosis Assay Kit also includes a green fluorescent cell viability dye, which allowing the simultaneous detection of both live cells and the process of phagocytosis by fluorescent microscopy. This assay can also be adapted for fluorescence micro-plate reader and flow cytometry detections.

Platform

Fluorescence microscope

Recommended plate	Black wall/clear bottom
Instrument specification(s)	Texas Red/FITC filter

Components

Example protocol

AT A GLANCE

Protocol Summary

Plate cells
Add 12.5 µL Protonex™ 600 Red-Latex Beads Conjugate
Incubate at 37 °C for 4 hours
Add 12.5 µL CytoTrace™ Green
Incubate at 37 °C for 30 minutes
Monitor fluorescence by microscopy using Texas Red and FITC filters

Important Thaw all the kit components to room temperature before starting the experiment.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protonex™ 600 Red-Latex Beads Conjugate solution (12X)

Add 8 µL of Protonex™ 600 RedLatex Beads Conjugate (Component A) in 2 mL cell growth medium (containing 10% FBS).
Note The unused beads can be stored at 4 °C.

2. CytoTrace™ Green stock solution (400X)

Add 20 µL of DMSO (Component C) into the vial of CytoTrace™ Green (Component B) and mix them well.
Note The unused CytoTrace™ Green DMSO stock solution can be aliquoted into single use vials and stored at -20 °C.

PREPARATION OF WORKING SOLUTION

CytoTrace™ Green working solution (12X)

Add 5 µL of CytoTrace™ Green stock solution (400X) in 2 mL of cell growth medium and mix them well.

SAMPLE EXPERIMENTAL PROTOCOL

Add 25 µL of 6X Cytochalasin D as a positive control or your test compound into each well.
Note The concentration of Cytochalasin D used in the assay should be optimized for each individual cell line.
Incubate the plate in the cell incubator for 30 minutes.
Add 12.5 µL of Protonex™ 600 Red-Latex Beads Conjugate solution (12X) into each well.
Incubate the plate in the cell incubator for 4 hours.
Note The incubation time should be optimized by users for each individual cell lines.
Add 12.5 µL of CytoTrace™ Green working solution (12X) to each well.
Incubate the plate in the cell incubator for 30 minutes.
Wash the plate twice with 1X PBS.
Observe phagocytosis inside the cells with Texas Red filter (Ex/Em = 570/600 nm) and CytoTrace™ Green with FITC filter (Ex/Em = 490/525 nm).

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	576
Emission (nm)	597

Images

Figure 1. Examination of phagocytosis in RAW 264.7 cells using Cell Meter™ Fluorimetric Phagocytosis Assay Kit (Cat# 21225). RAW 264.7 cells were incubated with (B) or without (A) Cytochalasin D for 30 min before Protonex™ 600 Red-Latex Beads in growth medium was added and incubated for 4 hours before Cell Tracker was added and incubated for 30 minutes. The images were taken using Keyence Fluorescence microscopy.

Citations

View all 4 citations: Citation Explorer

Methylsulfonylmethane protects against lethal dose MRSA-induced sepsis through promoting M2 macrophage polarization
Authors: Ma, Wei and Ao, Shengxiang and Zhou, Jianping and Li, Jiaxin and Liang, Xin and Yang, Xue and Zhang, Hao and Liu, Boyang and Tang, Wanqi and Liu, Haoru and others,
Journal: Molecular Immunology (2022): 69--77

The N 6-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of STAT1 mRNA
Authors: Liu, Yihan and Liu, Zhujiang and Tang, Hao and Shen, Yicong and Gong, Ze and Xie, Nan and Zhang, Xu and Wang, Wengong and Kong, Wei and Zhou, Yuan and others,
Journal: American Journal of Physiology-Cell Physiology (2019): C762--C775

The N6-Methyladenosine (m6A)-Forming Enzyme METTL3 Facilitates M1 Macrophage Polarization through the Methylation of STAT1 mRNA
Authors: Liu, Yihan and Liu, Zhujiang and Tang, Hao and Shen, Yicong and Gong, Ze and Xie, Nan and Zhang, Xu and Wang, Wengong and Kong, Wei and Zhou, Yuan and others, undefined
Journal: American Journal of Physiology-Cell Physiology (2019)

PHD2 is a regulator for glycolytic reprogramming in macrophages
Authors: Guentsch, Annemarie and Beneke, Angelika and Swain, Lija and Farhat, Katja and Nagarajan, Shunmugam and Wielockx, Ben and Raithatha, Kaamini and Dudek, Jan and Rehling, Peter and Zieseniss, Anke and others, undefined
Journal: Molecular and Cellular Biology (2016): MCB--00236

References

View all 56 references: Citation Explorer

Monitoring phospholipid dynamics during phagocytosis: application of genetically-encoded fluorescent probes
Authors: Sarantis H, Grinstein S.
Journal: Methods Cell Biol (2012): 429

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock
Authors: Schreiner L, Huber-Lang M, Weiss ME, Hohmann H, Schmolz M, Schneider EM.
Journal: J Cell Commun Signal (2011): 135

The application of fluorescent probes for the analysis of lipid dynamics during phagocytosis
Authors: Flannagan RS, Grinstein S.
Journal: Methods Mol Biol (2010): 121

Quantification of microsized fluorescent particles phagocytosis to a better knowledge of toxicity mechanisms
Authors: Leclerc L, Boudard D, Pourchez J, Forest V, Sabido O, Bin V, Palle S, Grosseau P, Bernache D, Cottier M.
Journal: Inhal Toxicol (2010): 1091

Analysis of macrophage phagocytosis: quantitative assays of phagosome formation and maturation using high-throughput fluorescence microscopy
Authors: Steinberg BE, Grinstein S.
Journal: Methods Mol Biol (2009): 45

Phagocytosis and postphagocytic reaction of cord blood and adult blood monocyte after infection with green fluorescent protein-labeled Escherichia coli and group B Streptococci
Authors: Gille C, Leiber A, Mundle I, Spring B, Abele H, Spellerberg B, Hartmann H, Poets Ch F, Orlikowsky TW.
Journal: Cytometry B Clin Cytom (2009): 271

A fluorescently tagged C-terminal fragment of p47phox detects NADPH oxidase dynamics during phagocytosis
Authors: Li XJ, Tian W, Stull ND, Grinstein S, Atkinson S, Dinauer MC.
Journal: Mol Biol Cell (2009): 1520

Analysis of phosphoinositide dynamics during phagocytosis using genetically encoded fluorescent biosensors
Authors: Cosio G, Grinstein S.
Journal: Methods Mol Biol (2008): 287

Development of a highly specific rhodamine-based fluorescence probe for hypochlorous acid and its application to real-time imaging of phagocytosis
Authors: Kenmoku S, Urano Y, Kojima H, Nagano T.
Journal: J Am Chem Soc (2007): 7313

The nonopsonic allogeneic cell phagocytosis of macrophages detected by flow cytometry and two photon fluorescence microscope
Authors: Liu GW, Ma HX, Wu Y, Zhao Y.
Journal: Transpl Immunol (2006): 220