Protocol for flow cytometry on tissues

I. Required material
Reagents
- A primary antibody , against the target molecule
- A Fc receptor blocker : « Anti-CD16/32, block Fc binding » that non-specifically binds primary antibodies
- 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
- An intracellular fixation buffer
- Red Blood Cells Lysis buffer
- Trypan blue for counting cells

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


- 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 : 2 to 3 hours


III. Protocole
Preparation of cells
- Crush the tissue to obtain a cell suspension
- Transfer the result in a 50 mL tube and let big and fat debris sediment
- Centrifugate cell suspension during 5 min (300 - 400g) at 4°c and discard supernatant
- In case of blood presence, resuspend the pellet in 3 mL of lysis buffer during 10 mn at 37°c, dilute in 10 mL of PBS and recentrifugate suspension during 5 min (300 - 400g) at 4°C and discard supernatant
- Resuspend the pellet in 50 mL of assay buffer and count cells with 100 µl of this solution diluted in Trypan blue
- Centrifugate cell in suspension for 5 mn (300 - 400g) at +4°c and discard supernatant

Optionnal : For indirect staining, repeat the staining step with secondary antibody with the same protocol and similar observations
- Resuspend cells in reaction buffer to obtain a final concentration of 2 x 107 cells / ml

Preparation of antibody and incubation with cells
- 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.

- Ajouter 200 µl de tampon de réaction pour arrêter la réaction
- Centrifuger la suspension cellulaire pendant 5 min (300 - 400g) à 4°c et enlever le surnageant
- Répéter deux fois les lavages des cellules avec 200 µl de tampon de réaction

Optionnal : For indirect staining, repeat the staining step with secondary antibody with the same protocol and similar observations
- 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