Preparation of Peritoneal Macrophages, Bone Marrow, Thymus and Spleen Cells

Reagents

Phosphate buffered saline (BUF036A) containing 1% bovine serum albumin (PBS/BSA).

Ammonium chloride lysis buffer: 0.16M ammonium chloride, 0.17M Tris, pH7.2

Optional PBS/BSA with 25 μg/ml DNAse I or 5 mM EDTA to reduce cell aggregates

Methods

1. Prepare a single cell suspension from relevant tissue. 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 through a 40 μm cell strainer.
2. Centrifuge at 300-400 g for 5 minutes at 4^oC.
3. Discard supernatant and re-suspend pellet in 10 ml ammonium chloride lysis buffer.
4. Mix and incubate for 2 minutes at 4^oC. Do not exceed this time.
5. Centrifuge at 300-400 g for 5 minutes at 4^oC.
6. Discard supernatant and resuspend pellet in 10ml cold (4^oC) PBS/BSA
7. Centrifuge at 300-400 g for 5 minutes at 4^oC.
8. Discard supernatant and re-suspend pellet to a final volume of 10ml with cold (4^oC) PBS/BSA.
9. Count cells using a hemocytometer or an automated cell counter such as the TC20™ Automated Cell Counter (Bio-Rad product code 1450102).
10. Adjust suspension if necessary to give a final count of 0.7 – 1.2 x 10⁷/ml.

Notes

• The following should be considered when designing your Flow Cytometry experiments:
  • To avoid unspecific binding, you also need to block Fc receptors on cell types such as spleen cells, with FcR blocking reagents e.g. Bio-Rad’s Mouse Seroblock reagent (BUF041).
  • Appropriate controls should always be carried out, for flow cytometry the following should be considered for inclusion;
    • Isotype controls used to determine if the staining is specific.
    • Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
• For all multi-color Flow Cytometry experiments it is advisable to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.