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

Reagents

• Phosphate Buffered Saline 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Staining Buffer (BUF073)
• Erythrolyse Red Blood Cell Lysing Buffer, 10x (BUF04), dilute to 1x using distilled water
• Anticoagulant
  Note: For basic staining, any appropriate anticoagulant, such as heparin, ethylenediaminetetraacetic acid (EDTA), or acid citrate dextrose, may be used. In some instances, specific anticoagulants may be required.
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock FcR (BUF041)
• Live/dead dye
• Optional: Fixation Buffer (BUF071)

Method

1. Collect blood into an appropriate anticoagulant.
2. Aliquot 2–3 mL of whole blood into a 50 mL centrifuge tube.
3. Add 20–25 mL of PBS/BSA, prewarmed to 37°C, and mix well.
4. Centrifuge at 300–400 x g for 5 min at 37°C. Carefully aspirate the supernatant, taking care not to disturb the cell pellet, and resuspend the pellet in the residual supernatant.
5. Repeat steps 3 and 4 twice more for a total of three washes.
6. Add 2–3 mL cold (4°C) Staining Buffer.
7. Aliquot 100 μL of the cell suspension into as many 5 mL tubes as required.
8. Add directly conjugated antibody at the dilution determined from a titration experiment, or the vendor-recommended dilution. Mix well and incubate at 4°C for 30–60 min, avoiding direct light.
9. Add 2 mL Staining Buffer, centrifuge at 300–400 x g for 5 min, and discard the supernatant.
10. Resuspend in 2 mL of freshly prepared 1x Erythrolyse Red Blood Cell Lysing Buffer (BUF040). Incubate for 10 min at room temperature (RT). Centrifuge at 300–400 x g for 5 min and discard the supernatant.
11. Add 2 mL of RT PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard the supernatant.
12. Optional: Fix cells by resuspending in 200 μL Fixation Buffer. Incubate for 20 min at RT in the dark. Centrifuge at 300–400 x g for 5 min and discard the supernatant.
13. Resuspend cells in 200 μL cold (4°C) PBS and store in the dark at 4°C.
14. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hours.

Notes

• The cell washing procedure described above does not affect other cell surface antigens and has been shown not to affect the recovery of individual cell subsets
• It is best practice to block Fc receptors prior to antibody staining to avoid nonspecific binding. We recommend using Bio-Rad's Human Seroblock (BUF070) for blocking human cells and Bio-Rad's Mouse Seroblock FcR (BUF041) for blocking mouse cells
• Including a viability dye is recommended to allow gating on live cells during analysis. The most appropriate live/dead dye is dependent on the fluorophores used and whether the cells are to be fixed. For fixed cells, a fixable dye, such as VivaFix Cell Viability Assay (13511), must be used
• Staining can also be performed in 96-well plates. Adjust wash volumes to 200 μL
• All antibodies should be titrated prior to use to ensure the optimal concentration is used