Note: The detection of intracellular cytokines requires a cell permeabilization step prior to staining. The method described below has been found to provide excellent results in our hands; however, other permeabilization techniques have been published, and may also be successfully used in this application. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that 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.

General procedure:

1. Harvest cells following stimulation or other appropriate treatment (see below).

2. Determine total number of cells present, and wash twice in wash buffer.

3. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at this stage. Following staining wash cells once in PBS and discard supernatant.

4. Resuspend cells in Leucoperm™ solution A (Catalogue number BUF09), using 50ml per 5x10⁵ cells. Incubate for 15 minutes at room temperature. Wash once in wash buffer.

5. Resuspend cells in Leucoperm™ solution B, using 50ml per 5 x 10⁵ cells. Aliquot 50ml of cell suspension into the required number of tubes containing directly conjugated anti-cytokine antibody. Incubate for 30 minutes at room temperature.

6. Wash once in wash buffer, and resuspend in 0.25ml 0.5% paraformaldehyde in PBS. Store at 4^oC until acquisition on the flow cytometer, preferably within 24 hours.

Cell Stimulation

Resting cells often require stimulation in vitro prior to the detection of intracellular cytokines. The conditions used for this will be dependent upon the cell system being investigated, and the cytokines being studied. The technique outlined below is that used by us to stimulate the expression of IL-2, gIFN and TNFa in human peripheral blood mononuclear cells (PBMCs). We recommend that investigators establish their own protocols according to the experimental system that they are using.

1. Prepare PBMCs by density gradient centrifugation. Wash twice in PBS. Adjust cell density to 1 x 10⁶ cells/ml in RPMI containing 10% FCS.

2. Pipette 0.5ml of cell suspension into wells of a 24 well plate. Add 0.5ml of RPMI containing 10% FCS, 2mM ionomycin, 10ng/ml PMA and 6mM monensin.

3. Incubate for 10-12 hours at 37oC in 5% CO₂ incubator. Harvest cells.

Buffers

Wash buffer PBS containing 1% BSA and 0.1% sodium azide.

NOTES

This information and associated protocols should be of assistance in the design and implementation of experiments investigating intracellular antigens such as cytokines. However, it should be remembered that the successful application of these reagents and associated products is subject to a number of experimental variables, and therefore researchers should always determine optimal conditions for their own assay system.