Preparation of Peritoneal Macrophages, Bone Marrow, Thymus, and Spleen Cells for Flow Cytometry
Preparation of Peritoneal Macrophages, Bone Marrow, Thymus, and Spleen Cells for Flow Cytometry
Cells of interest may be from a tissue, such as the peritoneum, bone marrow, thymus, or spleen. To analyze these samples by flow cytometry, a single cell suspension must be generated followed by the removal of any contaminating red blood cells with a red cell lysis step. A certain level of technical skill and immunological knowledge is required for the successful implementation of these techniques; these are guidelines only and may need to be adjusted for particular tissue types or applications.
Flow cytometry analysis may be required from cells derived from other sources. For the preparation of cells from tissue culture please refer to Bio-Rad’s protocol FC1. Further to this, a protocol (FC2) is also available for the sample preparation of peripheral blood mononuclear cells.
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).
