Protocol of Direct Staining Flow Cytometry

Direct staining flow cytometry is one of the most common and simplest staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens. The provided protocols here are only guidelines and may need to be adjusted for particular applications. They mainly include the following parts:

• Protocol A: Protocol of Direct Immunofluorescence Staining Surface Epitopes of Cells and Blood
• Protocol B: Protocol of Direct Immunofluorescence Staining of Immunoglobulin Light Chains on B Lymphocytes in Whole Blood

Refer to our Troubleshooting of Direct Staining Flow Cytometry when you are in trouble. If necessary, please don’t hesitate to contact us for additional help.

Protocol A: Protocol of Direct Immunofluorescence Staining Surface Epitopes of Cells and Blood

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA
• Optional: 0.5% (w/v) paraformaldehyde in PBS

Methods

1. Prepare cells appropriately. Please refer to Cell Preparation for Flow Cytometry. 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 antibody at the vendor-recommended dilution. Mix thoroughly and incubate at 4°C for at least 30 minutes. Avoid direct light.
4. Wash cells with 2 ml of cold PBS-BSA. Centrifuge at 400 g for 5 minutes. Discard the supernatant.
5. Resuspend cells in 200 μl of cold PBS or with 200 μl of 0.5% paraformaldehyde in PBS if required.
6. Acquire data by Flow Cytometry. Analyze fixed cells within 24 hours.
7. 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.

Protocol B: Protocol of Direct Immunofluorescence Staining of Immunoglobulin Light Chains on B Lymphocytes in Whole Blood

The immunofluorescent staining of immunoglobulin (Ig) expression by B lymphocytes in whole blood requires a procedure to remove serum Ig which otherwise interfere and block staining with Ig-specific antibodies.

Reagents

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

Methods

1. Collect blood into appropriate anticoagulant such as heparin, acid citrate dextrose and EDTA.
2. Aliquot 2-3 ml of whole blood into a 50 ml centrifuge tube.
3. Add 20-25 ml of PBS-BSA, pre-warmed to 37°C. Mix thoroughly.
4. Centrifuge at 400 g for 5 minutes at 37°C. Carefully remove the supernatant. Resuspend the pellet in the residual supernatant.
5. Repeat step 3 and step 4 twice more for a total of three washes.
6. Resuspend in 2-3 ml cold PBS-BSA. Aliquot 100 μl of the washed blood into the required number of test tubes.
7. Add appropriate volume of antibody at the vendor-recommended dilution and incubate at 4°C for at least 30 minutes. Avoid direct light.
8. Add 2 ml of freshly prepared red cell lysing buffer. Mix thoroughly.
9. Incubate for 10 minutes at RT.
10. Centrifuge at 400 g for 5 minutes. Remove the supernatant.
11. Wash with 2 ml of PBS-BSA. Centrifuge at 400 g for 5 minutes at RT. Remove the supernatant.
12. Resuspend cells in 200 μl cold PBS or with 200 μl of 0.5% paraformaldehyde in PBS if required.
13. Acquire data by Flow Cytometry. Analyze fixed cells within 24 hours.
14. Data analysis.

Notes

1. The cell washing procedure described above has no effect on other cell surface antigens and the recovery of individual cell subsets.
2. In order to avoid non-specific binding, you need to block FcR on cell types such as spleen cells with FcR blocking reagents.
3. 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.
4. 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.