Direct staining of intracellular antigens: Leucoperm Accessory Reagent method

Reagents

Leucoperm Accessory Reagent (Product code BUF09). Includes Reagent A (cell fixation agent) and Reagent B (cell permeabilization agent).

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

PBS.

Erythrolyse red blood cell lysing buffer (BUF04).

Anticoagulant (Note: for basic staining any appropriate anticoagulant, such as heparin, EDTA, or acid citrate dextrose, may be used. In some instances specific anticoagulants may be required)

Optional: 0.5% (w/v) paraformaldehyde in PBS (Note: dissolve on heated stirrer and cool before use)

Method

1. Harvest cells after appropriate treatment and determine the total number present. Adjust cell suspension to a concentration of 1 x 10⁷ cells/ml with cold (4^oC) PBS/BSA.
   [Whole blood samples may be used undiluted unless the cell count is high, as in leukemia samples. Use appropriate anticoagulant]
2. Add 100 μl of cell suspension [or whole blood] to the appropriate number of test tubes.
3. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at 4^oC, avoiding direct light.
4. Wash cells once in 2ml cold (4^oC) PBS/BSA and discard the supernatant.
5. Re-suspend cells in 100 ul cold (2-8^oC) Leucoperm Reagent A (cell fixation agent) per 1 x 10⁶ cells. Incubate for 10 minutes at 2-8^oC.
6. Add 3ml room temperature PBS/BSA and centrifuge for 5 minutes at 300-400 g at room temperature.
7. Remove supernatant and add 100 μl Leucoperm Reagent B (cell permeabilization agent) per 1 x 10⁶ cells. Add the directly conjugated antibody at the vendor-recommended dilution and incubate at 4^oC for at least 30 minutes, avoiding direct light.
   [To the blood suspension add 2 ml freshly prepared erythrolyse red cell lysing buffer and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 300-400 g for 5 minutes and discard the supernatant. Wash with 2ml room temperature PBS/BSA, centrifuge at 300-400g for 5 minutes at room temperature and discard the supernatant. Continue to step 8.]
8. Wash once in PBS and then re-suspend in 200 μl cold (4^oC) PBS for immediate analysis or with 200 μl of 0.5% formaldehyde in PBS if required.
9. Acquire data by Flow Cytometry. Analyze fixed cells within 24 hours.

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.