Note: Phycoerythrin conjugates are not suitable for the detection of cell surface antigens using this method due to damage of the RPE at low temperatures.
The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described below has been found to provide excellent results in our hands; however other permeabilization techniques have been published, and may also be successfully used in this application. This modification is particularly suitable for the detection of some nuclear antigens, such as PCNA and Ki67. In some cases specific recommendations are provided on product datasheets, and this method 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.
Leucoperm (BUF09) Wash Buffer, Phosphate Buffered Saline (PBS) containing 1% PBS and 0.09% Sodium azide.
- 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.]
- Add 100 μl of cell suspension [whole blood] to the appropriate number of test tubes.
- 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.
- Resuspend cells in cold (2-8°C) LeucopermTM Reagent A using 100 μl per 1 x 106 cells. Incubate for 10 minutes at 2-8°C.
- Add 500 μl of ice cold absolute methanol, vortex and incubate for 10 minutes at 2-8°C.
- Add 3ml PBS and centrifuge for 5 minutes at 300 g.
- 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.
- 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.]
- Acquire data by flow cytometry. Analyse fixed cells within 24 hours.