Protocol of Indirect Staining Flow Cytometry

This protocol is applicable when using unconjugated or biotin-conjugated monoclonal antibody (mAb) and polyclonal antibody (pAb) recognizing cell surface antigens. A conjugated secondary reagent must be used to visualize the primary antibody. This method only provides a general procedure for use when performing your indirect staining of cells and blood for flow cytometry. In some cases, the protocol may need to be adjusted for particular applications. Refer to our Troubleshooting of Indirect Staining Flow Cytometry when you get in trouble. Please contact us for additional help if necessary.

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA
• Red blood cell lysing buffer
• Anticoagulant
• Optional: 0.5% (w/v) paraformaldehyde in PBS

Methods

1. Prepare cells appropriately. Refer to Cell Preparation for Flow Cytometry and adjust the cell suspension to a concentration of 1 x 10⁷ cells/mL with cold PBS-BSA.

2. Aliquot 100 μL of the cell suspension into required number of tubes.

3. Add primary antibody at the vendor-recommended dilution. Mix thoroughly and incubate at 4°C for at least 30 minutes.

4. Wash cells with 2 mL of cold PBS-BSA. Centrifuge at 400 g for 5 minutes and remove the supernatant.

5. Add an appropriate secondary reagent at the vendor-recommended dilution. Mix thoroughly and incubate at 4°C for at least 30 minutes. Avoid direct light.

6. Centrifuge at 400 g for 5 minutes at RT. Discard the supernatant.

7. Resuspend cells in 200 μL of cold PBS or with 200 μL of 0.5% paraformaldehyde in PBS if required.

8. Acquire data by flow cytometry.

9. Data analysis.

Notes

1. In order to avoid non-specific binding, you need to block FcR on cell types such as spleen cells with FcR blocking reagents.
2. Appropriate controls should always be carried out. For flow cytometry, the following should be considered for inclusion
• Isotype controls used to determine if the staining is specific.
• Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
3. For all multi-color flow cytometry experiments, it is suggested to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.