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

Bio-Rad

Flow Cytometry

This technique is applicable when using unconjugated or biotin-conjugated monoclonal and polyclonal antibodies recognizing cell surface antigens. A conjugated secondary reagent must be used to visualize the primary antibody, e.g. streptavidin in the case of biotin. Please note that RPE conjugates should be handled in the dark throughout.

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:


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


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 primary antibody at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.
  4. Add 2 ml of PBS/BSA buffer, centrifuge at 400 g for 5 minutes and discard the resulting supernatant.
  5. Add an appropriate secondary reagent at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.
  6. Wash cells with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the supernatant.
    [To the blood suspension add freshly prepared red cell lysis buffer, e.g. 2 ml of AbD Serotec’s Erythrolyse BUF04
  7. 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.]
  8. Resuspend cells in 0.2 ml of PBS/BSA or with 0.2 ml of 0.5% paraformaldehyde in PBS/BSA if required.
  9. Acquire data by flow cytometry. Analyze fixed cells within 24 hours.

Appropriate standards should always be included, e.g. an isotype-matched control sample. It may also be useful to include a control in which no primary antibody is used at all, to determine any non-specific binding of the secondary reagent to the target cells.

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