Flow cytometric surface staining procedure
Sample preparation
1. Collect cells, filter through a 200 mesh sieve, collect the filtrate, centrifuge at 300 g for 5 min, discard the supernatant.
2. Add appropriate amount of cell staining buffer (or PBS containing 1% BSA) to the cells, and resuspend the cells by gently blowing with a pipette gun.
Cell counting
After counting the suspension with a blood counting plate or other instrument, adjust the cell concentration to approximately 1 × 107/mL
Setting up experimental groups
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Sequestering Fc Receptors
Closure of the Fc receptor reduces non-specific staining during the staining process.
For mouse samples, purified CD16/CD32 monoclonal antibody binds to FcγRIII/II, sealing off non-specific staining and reducing the background fluorescence of negative cells to the level of unlabeled cells. Add 0.5~1μg of pure anti-mouse CD16/32 monoclonal antibody and incubate for 10 minutes at room temperature.
For rat samples, blocking can be performed with an excess of purified Ig of the same source and isoform as the fluorescent antibody or serum of the same source, or with a commercial Fc receptor blocker.
For human samples, purified CD16 monoclonal antibody can be used as a blocking FCR blocker. Add 1 μg of pure anti-human CD16 monoclonal antibody and incubate for 10 min at room temperature.
Cell staining
1. Add fluorescent labeling antibody according to the recommended dosage in the instruction manual, mix well and incubate at 4℃ for 30 minutes, protected from light.
2. Add cell staining buffer (or PBS containing 1% BSA) and resuspend the cells. Centrifuge the cell suspension at 300g for 5 minutes and discard the supernatant.
3. Add 200 μL of cell staining buffer (or PBS containing 1% BSA) to resuspend the cells, which were detected and analyzed by flow cytometry.
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