Cell subsets can be identified based on the expression of cell surface antigens, this is known as immunophenotyping. This protocol describes indirect staining, which is a two-step staining procedure. Firstly, cells are incubated with unconjugated or biotin-conjugated monoclonal or polyclonal antibodies recognizing cell surface antigens. A second step is then performed using a conjugated secondary reagent to visualize the primary antibody, for example, streptavidin in the case of biotin.
This method provides a general staining procedure for use with Bio-Rad reagents. 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 antibody vial. A certain level of technical skill is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular cell types or applications. Specific methodology for whole blood appears in [ ] brackets.
Note: for basic staining any appropriate anticoagulant, such as heparin, EDTA, or acid citrate dextrose, may be used. In some instances, specific anticoagulants may be required
1. Prepare cells appropriately. For information on preparation of cells from tissue culture cell lines, tissues, and isolation of peripheral blood mononuclear cells from whole blood refer to protocol FC1, FC2, or FC3. Resuspend cells at a concentration of 1 x 107 cells/ml in Staining Buffer (BUF073). [Undiluted whole blood in an appropriate anticoagulant].
2. Aliquot 100 μl of the cell suspension [or whole blood] into as many 5 ml tubes as required.
3. Add unconjugated or biotin labeled primary antibody at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at 4oC for 30–60 min.
4. Add 2 ml Staining Buffer (BUF073), centrifuge at 300–400 g for 5 min, and discard the supernatant.
5. Resuspend cells in 2 ml Staining Buffer (BUF073), centrifuge at 300–400 g for 5 min, and discard the supernatant. [Alternatively, for whole blood perform a red cell lysis. Resuspend in 2 ml of 1x Erythrolyse Red Blood Cell Lysing Buffer (BUF040). Incubate for 10 min at room temperature (RT). Centrifuge at 300–400 g for 5 min and discard the supernatant. Wash with 2 ml PBS/BSA, centrifuge at 300–400 g for 5 min, and discard the supernatant].
6. Resuspend cells in 100 µl Staining Buffer (BUF073). Add an appropriate conjugated secondary reagent at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at 4oC for at 30–60 min, avoiding direct light.
7. Centrifuge at 300–400 g for 5 min at RT and discard the supernatant.
8. Resuspend cells in 200 µl Staining Buffer (BUF073), centrifuge at 300–400 g for 5 min, and discard the supernatant.
9. Optional. Fix cells by resuspending in 200 µl fixation buffer (BUF071) for 20 min at RT in the dark, centrifuge at 300–400g for 5 min, and discard the supernatant.
10. Resuspend cells in 200 μl cold (4oC) PBS and store in the dark at 4oC.
11. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hours.