Direct immunofluorescence staining of intracellular antigens: Methanol plus 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 and determine the total number present. Adjust cell suspension to a concentration of 1 x 10⁷ cells/ml in PBS/BSA.
   ‎[Whole blood samples may also be used. Bio-Rad recommends the use of EDTA anti-coagulant in ‎these circumstances, although satisfactory results may be obtained using heparin or acid-citrate ‎dextrose.] ‎
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 μl 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 500 μl of ice cold absolute methanol, vortex and incubate for 10 minutes at 2-8^oC.
7. Add 3 ml cold (4^oC) PBS and centrifuge for 5 minutes at 300-400 g at 4^oC.
8. 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 2ml freshly prepared erythrolyse red cell lysing buffer and mix well. Incubate for 10 min at room temperature. Centrifuge at 300-400 g for 5 min and discard the supernatant. Wash with 2ml room temperature PBS/BSA, centrifuge at 300-400 g for 5 min at room temperature and discard the supernatant. Continue to step 9.]
9. Resuspend cells in 2 ml cold (4^oC) PBS and centrifuge at 300-400 g at 4^oC. Discard supernatant.
10. Resuspend cells in 200 μl cold (4^oC) PBS for immediate analysis or with 200 μl 0.5% formaldehyde in PBS if required.
11. Acquire data by flow cytometry. Analyze fixed cells within 24 hours.

Notes

• Phycoerythrin conjugates are not suitable for the detection of cell surface antigens using this method due to damage of the RPE at low temperatures.
• 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.