Flow Cytometry (F) Protocol for the Detection of Transcription Factors

A. Solutions and Reagents

NOTE: Prepare solutions with RODI (reverse osmosis deionized) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
2. Formaldehyde (methanol free).
3. 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).
4. Triton X-100.
5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: Immunostaining of surface antigens should be performed on live cells prior to fixation/permeabilization, as per manufacturer’s requirements.

1. Adjust cell suspension of freshly isolated cells to 1–2 x 10⁶ cells in 100 μl Incubation Buffer per assay tube.
2. Add antibodies against surface antigens to the assay tubes as per manufacturers’ recommended volume or concentration and incubate for 30 min on ice.
3. Add 2 ml of Incubation Buffer and wash by centrifugation.
4. Aspirate supernatant and resuspend in 500 μl of 2% formaldehyde.
5. Fix for 15 min at room temperature.
6. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

1. Add 1ml of 0.1% Triton X-100 (v/v in PBS) to the cell pellet.
2. Resuspend and let stand for 30 min at room temperature.
3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

1. Resuspend cell pellets in 100 μl of antibody working solution, diluted according to individual antibody datasheet or product webpage in Incubation Buffer.
2. Incubate for 1 hr at room temperature.
3. Wash 2X by centrifugation in Incubation Buffer.
4. If using a fluorochrome-conjugated primary antibody, resuspend cells in 350 μl of Incubation Buffer and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to Step 5.
5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer at the recommended dilution.
6. Incubate for 30 min at room temperature.
7. Wash 2X by centrifugation in Incubation Buffer.
8. Resuspend cells in 350 μl of Incubation Buffer and analyze on flow cytometer.