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

Reagents

• Phosphate buffered saline (catalog BUF036A) containing 1% bovine serum albumin (PBS/BSA)
• Erythrolyse Red Blood Cell Lysing Buffer (10x) (BUF04)
• dH2O
• Blocking reagent e.g., 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 from relevant tissue.

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.  

2. Centrifuge at 300–400 g for 5 mins at 4°C.
3. Discard supernatant and resuspend pellet in 10 ml 1x Erythrolyse Red Blood Cell Lysis Buffer
(dilute 10x solution 1 in 10 with dH2O).
4. Mix and incubate for up to 10 min at room temperature. Do not exceed this time.
5. Centrifuge at 300–400 g for 5 min at 4°C.
6. Discard supernatant and resuspend pellet in 10 ml cold (4°C) PBS/BSA
7. Centrifuge at 300–400 g for 5 min at 4°C.
8. Discard supernatant and resuspend pellet to a final volume of 10 ml with cold (4°C) PBS/BSA.
9. Adjust cell concentration to 1 x 10⁷ cells/ml.

Notes

• It is recommended that all containers which 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 to avoid nonspecific binding. To block mouse cells we recommend using Bio-Rad’s Mouse Seroblock FcR (BUF041).