Materials:
- Flow Buffer (PBS+2%FBS)
Protocol:
- Start with 100 ul of anti-coagulated whole blood. Add desired amount/concentration of purified primary antibodies to whole blood and incubate at room temperature for 30 min in the dark.
- Use Red Blood Cell (RBC) Lysis Buffer (e.g BioLegend Cat. #420301) and dilute to 1X working concentration with deionized water. Warm to room temperature before use. Add 2 ml of 1X RBC lysis buffer to blood/antibody solution and incubate at room temperature for 15 minutes. Centrifuge cells at 350 x g for 5 minutes and aspirate.
- After incubation, wash each sample with 1 ml flow buffer. Centrifuge for 3 minutes at 350 x g for 5 minutes and aspirate buffer.
- If using a primary antibody, resuspend each pellet in 200 ul flow buffer. Add secondary antibody at desired concentration/dilution (e.g. 1:50 dilution or 2 ug total). Incubate 30 min in the dark. If using a direct-labeled fluorescent antibody, proceed to step #6.
- Repeat wash step #3 for a total of two times.
- Resuspend cells in 200-500 ul flow buffer.
- Transfer cells to the appropriate tubes for flow analysis.