1) Prepare spleen, lymph node or T cell clone cells as single cell suspension, using erythrocyte lysis (water) or Lymphopaque as appropriate.
2) Stimulate cells for 2 hours in vitro with 50 ng/ml PMA (Sigma, P8139) + 500 ng/ml ionomicin (Sigma I0634) in phenol red free RPMI 1640 medium + 10% fetal calf serum at 37 deg C.
3) Add Brefeldin A (Sigma, B7651) to 10mg/ml for a further 2 hr at 37 deg C. Harvest cells and wash into PBS + 0.1% BSA + 0.1% NaN3.
4) Perform any antibody staining of the cell surface that is sensitive to fixation at this stage. If you are using cells with high levels of Fc receptors, and entirely non-antiglobulin based detection as below it is advisable to use 'Fc block' (anti-FCgRII/III: 2.4G2; Pharmingen 01241A).
5) Wash cells and fix with 2% v/v formaldehyde in PBS (20min, 4 deg C).
6) Wash cells and permeabilized with PBS + 0.5% saponin (Sigma S-2149).
7) We suggest the following antibody conjugates (usually at 10mcg/ml or as manufacturers recommendations) to detect mouse cytokines, added in saponin buffer for 30 mins at 4 deg C. I have grouped them in compatible pairs for multicolour analysis:
8) Wash extensively in saponin buffer followed by PBS + 0.1% azide + 1% BSA, + 5% heat inactivated normal rabbit serum (HINRS).
9) Label with antibodies to surface molecules that are resistant to fixation (30 mins 4 deg C) eg.
If you do not have a three or four colour FACS machine then different combinations of conjugates can obviously be used as appropriate.
10) Wash and then fix the cells by resuspending in PBS 1% BSA and adding an equal volume of 4% Formalin in PBS.
11) Note that these conditions of stimulation, staining and analysis were such that normal, naive CBA/Ca CD4+ spleen cells should be essentially negative for all above cytokine stains. This control negative staining with the actual conjugates is, in our view, much more useful than staining with isotype matched conjugated controls that may have different properties with regard to degree of conjugation, aggregation, stickiness etc.
Steve Cobbold 29th July 1998