Protocol: Direct staining of intracellular antigens by Flow Cytometry

For the detection of cell cycle antigens such as Ki-67, PCNA and BrdU, methanol modification is recommended – see protocol F5

The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described below produces excellent results in our hands; however, other permeabilization techniques have been published, and may also be used successfully for this application.

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. ‎

Note: Specific methodology for blood appears in [ ] brackets.

Reagents

1. Leucoperm (BUF09)

2. Wash Buffer
Phosphate Buffered Saline (PBS) containing 1% PBS and 0.09% Sodium azide.

1. Harvest cells and determine the total number present. Adjust cell suspension to a concentration of 1 x 107 cells/ml in PBS containing 1% BSA.
   [Whole blood samples may also be used. AbD Serotec recommends the use of EDTA anti-coagulant in these circumstances, although satisfactory results may be obtained using heparin or acid-citrate dextrose.]
2. Add 100 μl of cell suspension/whole blood to the appropriate number of test tubes.
3. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at this stage. Following staining, wash cells once in PBS/BSA and discard the supernatant.
4. Resuspend cells in LeucopermTM Reagent A (cell fixation agent) using 100 μl per 1 x 106 cells. Incubate for 15 minutes at room temperature.
5. Add 3ml PBS and centrifuge for 5 minutes at 300 g.
6. Remove supernatant and add 100 μl LeucopermTM Reagent B (cell permeabilization agent) per 1 x 106 cells and add 10 μl of the appropriate, directly conjugated antibody.
7. Vortex at low speed for 1-2 seconds and incubate for 30 minutes at room temperature.
8. Wash once in wash buffer, and then resuspend in sheath fluid for immediate analysis or with 0.25 ml of 0.5% formaldehyde in PBS/BSA if required.
   [To the blood suspension add freshly prepared red cell lysis buffer e.g. 2 ml of AbD Serotec’s Erythrolyse BUF04 and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant]
9. Acquire data by flow cytometry. Analyse fixed cells within 24 hours.