Introduction:
Cell surface markers can be used to define cell subsets based on developmental stage and analyzed by flow cytometry. These surface markers have different forms and functions. For example, CD4 is a surface marker for T helper cells that can be further differentiated based on expression of other chemokine receptors and cluster of differentiation (CD) markers. Live cells stained with antibodies can be sorted based on unique staining patterns and used for additional experiments.
1. Prepare cells
More detail you can view Sample Preparation for Flow Cytometry.
1) Collect cells, filtere through a 200-mesh sieve and collect the filtrate.Centrifuge at 300 × g for 5 min, and discard the supernatant.
2) Add cell staining buffer (or PBS with 0.1% BSA) to resuspend the sample.
2. Cell Counting
After counting the suspension with a hemocytometer or other instruments, adjust the cell concentration to about 1 × 107/mL.
3. Set Sample and Control
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Groups
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Tubes
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Controls
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Blank
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Single staining control
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Isotype control
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FMO
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Biological control
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Sample
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Experimental sample
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4. Block Fc Receptor
Block Fc receptors may reduce nonspecific immunofluorescent staining.
For human cells, EasyStain™ Human Fc Receptor Blocking Solution (E-CK-A171) can be used as an FcR blocking reagent. Add 5 μL of EasyStain™ Human Fc Receptor Blocking Solution, mix well, and incubate at room temperature for 10 min.
For mouse cells, Purified Anti-Mouse CD16/CD32 Antibody specific for FcγR III/II can be used to block nonspecific staining of antibodies, and reduces the background fluorescence of negative cells to the level of unlabeled cells. Add 1 μg of Purified Anti-Mouse CD16/32 Antibody (E-AB-F0997A) and incubate at room temperature for 10 min.
For rat cells, excessive purified Ig from the same source and subtype as fluorescent antibodies or serum from the same source can be directly used for blocking, or commercial FcR blocking agents can be used.
5. Cell Surface Staining
1). Add the antibody according to the recommended dosage of the instructions and mix well.
2). Incubate at 4°C for 30 min in the dark.
6. Detection
1). Add cell staining buffer, centrifuge at 300 × g for 5 min, and discard the supernatant.
2). Add 200 μL cell staining buffer to resuspend the sample.
3). Adjust instrument parameters,detection.
