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Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Green Fluorescence*
Product key features
- Utilizes unique Protonex™ Green 500-Latex Beads for enhanced detection
- Exhibits minimal background due to the fluorogenic properties of Protonex™ Green 500-Latex Beads
- Convenient Ex/Em wavelength well matches the standard FITC filter set
- Includes a red fluorescent cell viability dye for accurate live cell detection
- Suitable for various applications including microscopy, microplate assays, and flow cytometry
Product description
Cell Meter™ Fluorimetric Phagocytosis Assay Kit uses the unique Protonex™ Green 500-based latex bead conjugate. Protonex™ Green 500 bead conjugate is a unique pH probe that can be delivered into cells by phagocytosis. The bead conjugate is a ready to use suspension. Unlike most of the existing fluorescent dyes, the Protonex™ Green 500-latex bead conjugate is non-fluorescent. However, its fluorescence dramatically increases as the bead conjugate gets into acidic phagosomes/phagolysosomes. The spectral properties of Protonex™ Green 500 are similar to those of FITC, allowing for the use of common FITC filter sets in assays that use Protonex™ Green 500-latex bead conjugates. It provides a robust tool to study phagocytosis and its regulations. Cell Meter™ Fluorimetric Phagocytosis Assay Kit also includes a red fluorescent cell viability dye, which allows the simultaneous detection of both live cells and the process of phagocytosis by fluorescent microscopy. This assay can be adapted for either fluorescence microplate reader or flow cytometry detection. Phagocytosis is the cellular uptake of particles within a plasma-membrane envelope by a cell. The process is critical in the innate immune response by engulfment and destruction of invading microorganisms. Phagocytosis is required in maintaining tissue homeostasis and remodeling by the clearance of apoptotic bodies. Once internalized, the phagosome fuses with lysosomes to form a secondary phagolysosome for digestion, resulting in progressive decrease of pH.
Example protocol
AT A GLANCE
Plate cells.
Add Protonex™ Green 500-Latex Bead Conjugate solution.
Incubate at 37 °C for 4 hours.
Add CytoTrace™ Red.
Incubate at 37 °C for 30 minutes.
Monitor fluorescence by microscopy using FITC and Texas Red filters.
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
Plate cells overnight in a growth medium at 20,000-50,000 cells/well/100 µL in a 96-well plate.
Note: For the RAW 264.7 cells used in this assay, we recommend plating 50,000 cells per well in 100 µL of medium in a 96-well plate and incubating them overnight. It is important to optimize the cell density for each cell line individually.
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
Add 10 µL of Protonex™ Green 500-Latex Bead Conjugates (Component A) to 2 mL of cell growth medium (containing 10% FBS).
Note: Unused beads can be stored at 4 °C.
Add 20 µL of DMSO (Component C) to the vial containing CytoTrace™ Red (Component B), and mix thoroughly.
Note: Unused 400X CytoTrace™ Red stock solution in DMSO can be aliquoted into single-use vials and stored at -20 °C, protected from light.
Add 18 µL of 10 mM Cytochalasin D (not provided) or 18 µL of DMSO to 3 mL of cell growth medium. The final concentration in the well will be 10 µM.
PREPARATION OF WORKING SOLUTION
Add 5 µL of CytoTrace™ Green stock solution (400X) to 2 mL of cell growth medium and mix well.
SAMPLE EXPERIMENTAL PROTOCOL
Add 25 µL of 6X Cytochalasin D (not provided) as a positive control or your test compound into each well. The final concentration in the well should be 10 µM.
Note: To prepare a 6X Cytochalasin D solution, add 18 µL of 10 mM Cytochalasin D to 3 mL of cell growth medium, and mix thoroughly. Be sure to optimize the Cytochalasin D concentration for each specific cell line used in the assay.
Incubate the plate in a cell incubator for 30 minutes.
Add 12.5 µL of the 12X Protonex™ Green 500-Latex Bead Conjugate solution to each well.
Incubate the plate in a cell incubator for 4 hours.
Note: The incubation time should be optimized by users based on the specific requirements of each cell line.
Add 12.5 µL of the 12X CytoTrace™ Green working solution to each well.
Incubate the plate in a cell incubator for 30 minutes.
Wash the plate twice with 1X PBS.
Monitor phagocytosis within the cells using the FITC filter (Ex/Em= 490/525 nm) for green fluorescence, and observe CytoTrace™ Red using the Texas Red filter (Ex/Em = 570/600 nm) for red fluorescence.
Product family
| Name | Excitation (nm) | Emission (nm) |
| Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Red Fluorescence* | 576 | 597 |
Product protocol