Protocol: Direct Immunofluorescence Staining of Whole Blood for Intracellular CD68: The Leucoperm Method

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Anticoagulant
  Note: Any appropriate anticoagulant, such as heparin, ethylenediaminetetraacetic acid (EDTA), or acid citrate dextrose, may be used. In some instances, specific anticoagulants may be needed
• Erythrolyse Red Blood Cell Lysing Buffer (10x) (BUF04), dilute to 1x using distilled water
• Blocking reagent: 10% serum diluted in PBS; choose species to match target cell type, e.g., Mouse Serum (C11SA), Rat Serum (C13SB, C13SD), or human serum
• Required when staining cell surface antigens:
  - Staining Buffer (BUF073)
• Leucoperm (BUF09)
• Vivafix fixable live/dead dye (1351111, 1351112, 1351113, 1351115, 1351116, 1351117, 1351118)
• Optional: Fixation Buffer (BUF071)

Method

1. Use 2 mL undiluted whole blood in an appropriate anticoagulant per sample.
   Note: This protocol describes volumes for 2 mL whole blood, enough for 20 samples. Adjust volumes depending on your experimental setup.
2. Add 40 mL of diluted Erythrolyse (BUF04), mix well, and incubate for 10 min at room temperature (RT).
3. Centrifuge at 300–400 x g for 5 min and discard supernatant.
4. Add 40 mL PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.
5. Resuspend cells in 2 mL PBS.
   Note: Set aside 100 µL for every control sample that does not require staining with a viability dye. Continue from step 8 for the control samples.
6. Add Vivafix Cell Viability Assay at the recommended dilution (1/500 dilution of the stock solution) and incubate for 30 min at RT while avoiding direct light.
7. Resuspend cells in 20–50 mL cold (4°C) PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.
8. Repeat step 7.
9. Resuspend cells in 2 mL 10% serum and incubate for 15 min before adding 100 μL to as many 5 mL tubes as required.
10. Optional: Perform staining of cell surface antigens and associated wash steps. Refer to protocol FC4 for more details.
11. Add 2 mL PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.
12. Resuspend cells in 100 μL PBS/BSA.
13. Add 100 μL Leucoperm (BUF09) Reagent A (fixation medium) and incubate for 15 min at RT.
14. Add 3 mL PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.
15. Add 100 μL of Leucoperm (BUF09) Reagent B (permeabilization medium) containing 10% serum. Immediately add the anti-CD68 antibody at the dilution determined from a titration experiment or the vendor-recommended dilution and incubate at RT for at least 30 min while avoiding direct light.
16. Add 2 mL cold (4°C) PBS/BSA, centrifuge at 300–400 x g for 5 min, and discard supernatant.
17. Resuspend cells in 2 mL cold (4°C) PBS/BSA, centrifuge at 300–400 g for 5 min, and discard supernatant.
18. Optional: Fix cells by resuspending in 200 µL Fixation Buffer (BUF071), incubate for 20 min at RT in the dark, centrifuge at 300–400 x g for 5 min, and discard supernatant.
19. Resuspend cells in 200 μL cold (4°C) PBS and store in the dark at 4°C.
20. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

• Bio-Rad has several directly conjugated anti-CD68 antibodies suitable for flow cytometry. These include Anti-Rat CD68 (MCA341), Anti-Mouse CD68 (MCA1957), and Anti-Human CD68 (MCA6014, MCA5709, MCA2375) Antibodies
• The same protocol can also be used when staining peripheral blood mononuclear cells isolated from whole blood. Follow the protocol from step 5 and use 1 x 10⁶ cells for every sample required
• An unconjugated primary antibody can also be used. Add the primary antibody and wash (as per steps 15 and 16), and repeat steps 15 and 16 using an appropriate secondary antibody. There is no requirement to add additional Leucoperm
• It is important that appropriate controls are included. For example, staining unpermeabilized cells with anti-CD68 can be used to confirm the presence of a specific signal
• All antibodies should be titrated prior to use to ensure the optimal concentration is used