Pro-Apoptotic Factor Intracellular Labelling Protocol

1. Pelleted cells fixed with 0.25% paraformaldehdye for 1 hour on ice.
2. Cells were washed in PBS.
3. Primary mcab (1 mg) was added to cell pellets permeabilised in 0.01% saponin (Sigma) and incubated on ice for 30 mins.
4. Cells were washed in PBS/0.01% Saponin.
5. Secondary antibody (0.5 mg) were added to cell pellets and incubated for 30 mins on ice.
6. Cells were washed in PBS/0.01% Saponin and resuspended in 0.5 ml PBS.
7. At least 10,000 events were analysed by flow cytometry.