Protocol: Direct immunofluorescence staining of cells and blood for Flow Cytometry

This method provides a general procedure for use with the majority of AbD Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with the 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:

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PBS/BSA Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin

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Method:

 

1. Prepare cells appropriately. Adjust the cell suspension to a concentration of 1 x 107 cells/ml with PBS/BSA buffer.
   [Whole blood samples may be used undiluted unless the cell count is high, e.g. as in leukemia. EDTA and heparin are preferred anti-coagulants].
2. Aliquot 100 µl of the cell suspension [whole blood] into as many test tubes as required.
3. Add antibody at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.
4. Wash cells with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the resulting supernatant.
   [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. Wash with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the supernatant.]
5. Resuspend cells in 0.2 ml of PBS/BSA or with 0.2 ml of 0.5% paraformaldehyde in PBS/BSA if required.
6. Acquire data by flow cytometry. Analyze fixed cells within 24 hours.

Appropriate standards should always be included, e.g. an isotype-matched control sample.