General Intracellular Cytoplasmic Target Flow Protocol
Protocol for Cytoplasmic Targets:
- Optional: Perform cell surface staining as described in the previous section.
- Fix the cells by adding 100 µL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
- Permeabilize cells by adding 100 µL of a permeabization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
- For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
- To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
- Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
- Centrifuge for 5 minutes at 400 RCF.
- Discard supernatant and re-suspend in 1 mL of staining buffer + 0.1% permeabilizer.
- Stain each sample at 1 µL/ 1 x 106 cells of primary antibody or 1-3 µL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate on ice for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
- Following the primary/conjugate incubation, add 2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 5 minutes at 400 RCF.
- Remove supernatant and re-suspend each sample in 2 mL staining buffer + 0.1% permeabilizer, repeat wash for 5 minutes at 400 RCF.
- If using a directly conjugated antibody, after the second wash, re-suspend cell pellet to a final volume of 500 µL per sample and proceed with flow analysis.