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 the whole blood or tissue (spleen, lymph node, thymus or bone marrow), add cell staining buffer (or PBS with 0.1% BSA) to prepare monoplast suspension.
For cells stimulated in vitro, suspend the cells with cell staining buffer (or PBS with 0.1% BSA) after stimulation, then proceed to the 2)step.
2) Fill up the tube with cell staining buffer (or PBS with 0.1% BSA), centrifuge at 300 g for 5 min at room temperature. Then discard supernatant.
2. Erythrocyte lysis
1) If you need to split red blood cells, such as the spleen or bone marrow, dilute the 10 x Red Blood Cell Lysis Buffer (Ammonium-Chloride-Potassium Lysing Buffer, ACK buffer ) with DI water to 1 x ACK buffer, and put it to room temperature. Then re-suspend the cells in 3 mL 1X ACK buffer and incubate at room temperature for 3-5 min.
If there is no need to split red blood cells,such as lymph node or thymus, go directly to the 3) step.
2) Add 10 mL cell staining buffer (or PBS with 0.1% BSA) to terminate the ACK lysis, centrifuge at 300 g for 5 min at room temperature. Discard supernatant.
3) Fill up the tube with cell staining buffer(or PBS with 0.1% BSA) to 15 mL, centrifuge at 300 g for 5 min at room temperature. Discard supernatant.
4) Cells counting. Add cells staining buffer (or PBS with 0.1% BSA) to make cells suspension at concentration of 1x107 /mL. Then aliquots 100 µL cell suspension into FACs tube.
3. Block Fc receptor:
Block Fc receptors may reduce nonspecific immunofluorescent staining.
For Mouse cells: purified Anti-Mouse CD16/CD32 antibody specific for FcγR III/II can be used to block nonspecific staining of antibodies. Thus, block Fc receptors by pre-incubating cells with 0.5-1µg Anti-Mouse CD16/CD32 in 100 µl volume for 10 min at room temperature.
For Human and Rat cells: Pre-incubate the cells with excess irrelevant purified Ig from the same species and same isotype as the antibodies used for immunofluorescent staining or serums from the same species as the antibody used.
4. Cell surface staining:
1) Add labeled fluorescent antibody according to the datasheet recommendation, and incubate at 4 ℃ for 30 min in the dark.
2) Re-suspend the cells by adding 5 mL cell staining buffer (or PBS with 0.1% BSA), centrifuge at 300 g for 5 min, then discard supernatant.
3) Add 0.5 mL cell staining buffer (or PBS with 0.1% BSA) to re-suspend the cells. Detect and analyze by flow cytometry.
1. Antibody-binding kinetics is temperature-dependent. Staining on ice may require longer incubation times.
2. Some antibodies may require non-standard incubation conditions that will be noted on the technical data sheet provided with the antibody.
3. Fixed or delayed analysis may reduce the fluorescence signals. In order to get better results, it should be analyzed immediately after dyeing.