Protocol for flow cytometry on whole blood

Protocol for flow cytometry on whole blood



I. Required material
Reagents
- A primary antibody , against the target molecule
- A secondary antibody in cases of indirect staining with a non labeled primary antibody
- A non-relevant control isotype with a similar label than primary antibody

Buffers
- A staining buffer
- A Red Blood Cells Lysis (RBC) buffer
- An intracellular fixation buffer
- Trypan blue for counting cells
- Gaines sheath fluid

Material
- Pipets and Pipet-aid
- Centrifugator
- Freezer
- Flow cytometer


II. Experimentation length
- 30 mn sample preparation and antibody dilutions
- 30 mn to 1 h of antibody incubation
- 30 mn to 1 h of samples reading with the flow cytometer (depends on number of samples, concentration of cell and the cytometer capacity)
- TOTAL : 3 to 4 hours


III. Protocol
- Dilute primary antibody and control isotype in 50 µl of assay buffer in order to respect the desired concentration optimized after antibody titration
- Incubate 50 µl of cell preparation in 50 µl of antibody preparation or control isotype in a 96-wells plate
- Incubate during 15-30 mn at 4°c in a dark room

Note : You need to optimize the incubation time of antibody in respect to their affinity with the antigen.

- Add 200 µl of assay buffer to stop the reaction
- Centrifugate cell in suspension for 5 mn (300 - 400g) at +4°c and discard supernatant
- Repeat twice the wash steps with 200 µl of assay buffer - Centrifugate cell suspension for 5 min (300 - 400g) at 4°c

Optionnal : For indirect staining, repeat the staining step with secondary antibody with the same protocol and similar observations
- Resuspend in 2 ml of RBC lysis buffer and incubate for 10 mn at room temperature
- Add 2 ml of assay buffer to stop the reaction
- Centrifugate cell suspension for 5 mn (300 - 400g) at 4°c and discard supernatant
- Repeat twice the wash steps in 2 ml of assay buffer
- Resuspend in 500 µl of assay buffer or fixation buffer supplemented with 2%of formaldehyde
- Read cells in a flow cytometer


IV. Search engines
- Primary antibodies
- Secondary antibodies