Direct Staining of Intracellular Antigens by Flow Cytometry – the paraformaldehyde/saponin method

For use with AbD Serotec’s directly-conjugated FITC monoclonal antibodies.

The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described below has been extensively published and prior to the development of commercial reagents was the method of choice for this technique. We continue to recommend this protocol for certain antibodies, especially those for which the fixation step prior to permeabilisation is particularly important.

In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

Reagents:

Wash buffer
Phosphate buffered Saline (PBS) containing 1% BSA.
4% paraformaldehyde
PBS containing 4 g/100 ml paraformaldehyde.
(Note that preparation of this solution may require heating and may take a significant period of time. Ensure solution is cooled before use)
PBS/0.1% saponin
PBS containing 0.1% (w/v) saponin (0.1g per 100ml)
  1. Harvest cells and determine total number present. Wash twice in PBS.
  2. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at this stage. Following staining, wash cells once in PBS and discard supernatant.
  3. Resuspend cells in 4% paraformaldehyde solution using 100 ul per 1×106 cells. Incubate for 20 minutes at room temperature. Wash once in PBS.
  4. Resuspend cells in PBS/0.1% saponin solution, using 100 ul per 1 × 106 cells. Incubate for 15 minutes at room temperature.
  5. Aliquot 50 ul of cell suspension into the required number of tubes containing directly conjugated antibody. Incubate for 30 minutes at room temperature.
  6. Wash once in PBS/0.1% saponin buffer, and resuspend in 0.25 ml 0.5% paraformaldehyde in PBS. Store at 4°C until acquisition on the flow cytometer, preferably within 24 hours.