Protocol for intracellular flow cytometry

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 Permeabilization Buffer
- An intracellular fixation buffer

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


II. Experimentation length
- Stimulation of cells (depends on the cell type)
- 2 to 3 hours of antibodies 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
- Prepare cells according to your cell adapted protocol
- Stain cells according to the general protocol recommandations for cells in suspension
- After first wash, fix cells with 100 µl of fixation buffer and vortex tubes
- Incubate for 20 mn at room temperature in a dark room
- Centrifugate for 5 mn (300 – 400g) at 4°c
- Add 1 ml of permeabilization buffer
- Centrifugate for 5 mn (300 – 400g) at 4°c
- Add 20 µl of diluted primary antibody at an optimal concentration (according to the manufacturer) of 0,5 to 0,6 µg / 106 cellules) in permeabilization buffer and vortex
- Incubate tubes for 20 mn at room temperature
- Add 1 ml of permeabilization buffer
- Centrifugate for 5 mn (300 – 400g) at 4°c
- Resuspend cells 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