Direct staining of intracellular antigens: Leucoperm Accessory Reagent method
Direct staining of intracellular antigens and cytokines by Flow Cytometry: Leucoperm method
Method for cell permeabilization required prior to intracellular staining using Leucoperm Accessory Reagent.
The detection of intracellular antigens requires a cell permeabilization step prior to staining. For the detection of cell cycle antigens such as PCNA and BrdU, methanol modification is recommended – see protocol FC8 (Direct staining of intracellular antigens by Flow Cytometry: Leucoperm plus methanol method).
The method described below produces excellent results in our hands; however, other permeabilization techniques have been published, and may also be used successfully for this application. These methods should always be used in conjunction with the product and batch specific information provided with each vial. 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. Note: Specific methodology for blood appears in [ ] brackets.
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
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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 (4oC) PBS/BSA.
[Whole blood samples may be used undiluted unless the cell count is high, as in leukemia samples. Use appropriate anticoagulant] - Add 100 μl of cell suspension [or whole blood] to the appropriate number of test tubes.
- If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at 4oC, avoiding direct light.
- Wash cells once in 2ml cold (4oC) PBS/BSA and discard the supernatant.
- Re-suspend cells in 100 ul cold (2-8oC) Leucoperm Reagent A (cell fixation agent) per 1 x 106 cells. Incubate for 10 minutes at 2-8oC.
- Add 3ml room temperature PBS/BSA and centrifuge for 5 minutes at 300-400 g at room temperature.
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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 4oC 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.] - Wash once in PBS and then re-suspend in 200 μl cold (4oC) PBS for immediate analysis or with 200 μl of 0.5% formaldehyde in PBS if required.
- Acquire data by Flow Cytometry. Analyze fixed cells within 24 hours.
Notes
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The following should be considered when designing your Flow Cytometry experiments:
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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).
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Appropriate controls should always be carried out, for flow cytometry the following should be considered for inclusion;
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Isotype controls used to determine if the staining is specific.
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Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
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- 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.
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