Protocol: Preparation of Peritoneal Macrophages, Bone Marrow, Thymus, and Spleen Cells for Flow Cytometry

Reagents

• Phosphate Buffered Saline, 10x (PBS) (BUF036A) containing 1% bovine serum albumin (PBS/BSA)
• Erythrolyse Red Blood Cell Lysing Buffer (10x) (BUF04); dilute to 1x using distilled water
• Blocking reagent: Mouse Seroblock FcR (BUF041)
• Optional: PBS/BSA with 25 μg/mL DNAse I or 5 mM EDTA to reduce cell aggregates

Method

1. Prepare a single-cell suspension.
   Note: This may involve mechanical disruption or enzymatic digestion to generate a single-cell suspension. Keep cells on ice to minimize cell death, which can lead to cell aggregation. Addition of DNAse I or EDTA can also reduce aggregation. Large aggregates can be removed by passing the sample through a 40 μm cell strainer. Wash cells once in 2 mL cold (4°C) PBS/BSA and discard supernatant.
2. Centrifuge at 300–400 x g for 5 min at 4°C and discard supernatant.
3. Resuspend cells in 10 mL 1x Erythrolyse Red Blood Cell Lysis Buffer.
4. Mix and incubate for up to 10 min at room temperature.
   Do not exceed this time.
5. Centrifuge at 300–400 x g for 5 min at 4°C and discard supernatant.
6. Resuspend cells in 10 mL cold (4°C) PBS/BSA.
7. Centrifuge at 300–400 x g for 5 min at 4°C and discard supernatant.
8. Resuspend cells in 10 mL cold (4°C) PBS/BSA and adjust cell concentration to 1 x 10⁷ cells/mL.

Notes

• It is recommended that all containers that have come into contact with cells should be considered hazardous waste and discarded appropriately
• Ensure you prepare enough cells for your experiment. Typically, 10⁶ cells are used for each flow cytometry sample
• Count cells using a hemocytometer or an automated cell counter such as Bio-Rad’s TC20 Automated Cell Counter (1450102)
• It is best practice to block Fc receptors prior to antibody staining to avoid nonspecific binding. We recommend using Bio-Rad's Mouse Seroblock FcR (BUF041) for blocking mouse cells