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

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

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

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This method provides a general procedure for use with cell suspension cells acquired from the peritoneum, bone marrow, thymus and spleen. A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications.

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 4oC.
  3. Discard supernatant and re-suspend pellet in 10 ml ammonium chloride lysis buffer.
  4. Mix and incubate for 2 minutes at 4oC. Do not exceed this time.
  5. Centrifuge at 300-400 g for 5 minutes at 4oC.
  6. Discard supernatant and resuspend pellet in 10ml cold (4oC) PBS/BSA
  7. Centrifuge at 300-400 g for 5 minutes at 4oC.
  8. Discard supernatant and re-suspend pellet to a final volume of 10ml with cold (4oC) 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.

V1.1.2016