Flow Cytometry Protocol - Part 1: Cell Surface Staining
Step 1: Cell Counting
Collect the cultured cell into 15mL centrifuge tube and count. Centrifuge at 500g for 4min and remove supernatant.
Step 2: Single Cell Suspension Preparation
Resuspend the collected cell with 1mL 0.5%BSA/PBS solution. Centrifuge at 400g for 3min and remove supernatant; Repeat twice. Resuspend the cell with 1mL 0.5%BSA/PBS solution to achieve a concentration of 1x106 cell/mL. Add 100μl cell suspension into each well.
Step 3: Blocking Fc Receptors(Optional)
Incubate at room temperature for 10-20min.
Step 4: Primary Antibody Incubation
Add 100μl diluted primary antibody solution into each well, incubate at 2-8℃ for 30min.
Step 5: Washing
Centrifuge at 400g for 5min and remove supernatant. Add 200μl 0.5%BSA/PBS solution and resuspend. Centrifuge to remove supernatant. Repeat twice.
Step 6: Secondary Antibody Incubation
For non-fluorescent labeled antibodies, add 100μl fluorescent secondary antibody and resuspend. incubate at 2-8℃ for 30min in the dark area. For fluorescent labeled antibodies, jump to Step 8.
Step 7: Washing
Repeat Step 5.
Step 8: Cell Resuspension
Resuspend cell with 200μl 0.5%BSA/PBS solution. Store at 4℃ for further analysis.
Flow Cytometry Protocol - Part 2: Intracellular Staining
Step 1: Cell Counting
Collect the cultured cell into 15mL centrifuge tube and count. Centrifuge at 500g for 4min and remove supernatant.
Step 2: Single Cell Suspension Preparation
Resuspend the collected cell with 1mL 0.5%BSA/PBS solution. Centrifuge at 400g for 3min and remove supernatant; Repeat twice. Resuspend the cell with 1mL 0.5%BSA/PBS solution to achieve a concentration of 1x106 cell/mL. Add 100μl cell suspension into each well. Centrifuge at 400g for 5min and remove supernatant.
Step 3: Fixation
Add 100μl cell fixation buffer into each well to resuspend cell. Incubate at 2-8℃ for 20min.
Step 4: Washing
Centrifuge at 400g for 5min. Remove supernatant and add 200μl 0.5%BSA/PBS solution to resuspend. Centrifuge to remove supernatant. Repeat twice.
Step 5: Washing
Add 100μl cell permeabilization buffer into each well to resuspend cell. Incubate at room temperature for 20min.
Step 6: Washing
Repeat Step 4.
Step 7: Blocking Fc Receptors(Optional)
Incubate at room temperature for 10-20min.
Step 8: Primary Antibody Incubation
Add 100μl diluted primary antibody solution into each well, incubate at 2-8℃ for 30min.
Step 9: Washing
Repeat Step 4.
Step 10: Secondary Antibody Incubation
For non-fluorescent labeled antibodies, add 100μl fluorescent secondary antibody and resuspend. incubate at 2-8℃ for 30min in the dark area. For fluorescent labeled antibodies, jump to Step 11.
Step 11: Washing
Repeat Step 4.
Step 12: Experimental Analysis
Resuspend cell with 200μl 0.5%BSA/PBS solution. Store at 4℃ for further analysis.
REFERENCES
[1]Relevance of Antibody Validation for Flow Cytometry, PMID: 31577065.
[2]Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions, PMID: 32000909.