Suitable for use on single-cell suspensions from peripheral blood, lymphoid tissue or cultured cell-lines.
Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) to remove extracellular proteins, including cytokines.
Resuspend to 1-5 x 106 cells/mL in Wash Buffer.
Transfer 10-15 µL of the cell suspension to each reaction field on the adhesion slide.
Allow the cells to adhere electrostatically in a monolayer for 10 minutes at room temperature in the humidified chamber to prevent the cells from drying out.
Add 50 µL of ice-cold Fixation Buffer to each field to fix the cells.
Incubate for 20 minutes at 4°C.
Wash three times with Wash Buffer to remove free formaldehyde.
Add 25 µL of 2 fetal bovine serum in Wash Buffer to block unbound surface area on the slide.
Incubate for 10 minutes at 37°C.
Wash slides three times in Wash Buffer-Saponin. The slides are now ready for staining.
Alternatively, wash the slides in Wash Buffer and allow the slides to dry. Dried slides can be stored at -20°C several months before being stained. Prior to staining the slides should be washed in Wash Buffer-Saponin.
All antibody incubations and washes are performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes.
Detection using Biotin-labeled antibodies
Incubate in Endogenous Peroxidase Blocking Buffer for 30 minutes at room temperature in the dark to block endogenous peroxidase activity in the cells (this step can be omitted if cells are to be stained by fluorochromes or non-peroxidase based enzymatic methods).
Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30 minutes in the presence of saponin, described in steps 3-5.
Incubate each cell spot on slides with Avidin for 15 minutes supplemented with saponin (0.1 w/v).
Wash each cell spot on slides twice in Wash Buffer-Saponin.
Incubate in Biotin for 15 minutes supplemented with saponin (0.1 w/v).
Wash each cell spot on slides twice in Wash Buffer-Saponin.
Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL unlabeled or biotinylated cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin.
Wash slides three times in Wash Buffer-Saponin.
Note: If using R&D Systems biotinylated antibody skip steps 9 and 10 and continue.
Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab2 diluted 1:700; or biotin-goat anti mouse IgG1 or IgG2A or IgG2B diluted 1:500) in Wash Buffer-Saponin.
Wash slides three times in Wash Buffer-Saponin.
Cytokine-specific staining based on either biotinylated primary antibodies or unlabeled primary antibodies along with biotinylated secondary antibodies can then be developed by techniques based on immunoflourescence or immunoenzymatic methods.
Incubate slides for 30 minutes at room temperature with 15 µL of Oregon-Green Avidin or FITC-Avidin at a concentration of 2-5 µg/mL in Wash Buffer-Saponin.
Wash slides twice in Wash Buffer-Saponin.
Wash slides once in Wash Buffer only. Allow slides to air dry before mounting in Mounting Buffer. FITC staining, but not Oregon-Green staining, will require a mounting medium including some anti-fading substance to reduce UV quenching. It is possible to store fluorochrome-stained slides for long periods in the freezer, provided that they have not been mounted in Mounting medium.
Detection using fluorochrome-labeled antibodies
If cytokine producing cells are to be detected by fluorochrome-labeled primary or secondary antibodies there is no need to block endogenous peroxidase or biotin activity. Background signals are often reduced when fluorochrome-labeled antibodies are used in Wash Buffer-Saponin supplemented with 5 human AB serum.
Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL of either unlabeled or fluorochome-labeled primary cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin supplemented with 5 human AB serum.
Wash slides three times in Wash Buffer-Saponin.
Incubate cells for 30 minutes at room temperature with 15 µL of flurochrome-labeled secondary antibody (either FITC-labeled anti-mouse IgG1 or IgG2A or IgG2B diluted 1:300) in Wash Buffer-Saponin supplemented with 5 human AB serum.
Wash slides three times in Wash Buffer-Saponin and air dry the slides.
Mount and coverslip with Fluorescence Anti Fading Mounting Medium.
Question: Is the cytokine staining specific?
Test: Incubate cytokine-detecting antibody with target cytokine overnight and finally add 0.1 (saponin prior to staining as in Question 4.
Cause of Background: Cytokine-detecting antibody used at too high concentration or unsuitable for immunostaining.
Remedy: Titrate antibody or try another cytokine detecting antibody.
Permeabilization:
It is crucial that saponin is present during all antibody incubations and washes to make the staining procedure successful. For detection of intracellular cytokines, the cytokine-specific antibodies must penetrate through the cell surface membrane, the cytosol, the membranes of the endoplasmic reticulum and the Golgi organelle. The detergent, saponin has been shown to intercalate in the membranes to replace cholesterol and to permeabilize cells in a reversible way, maintaining much of the morphology of the membrane structure of the cell.
Fixation:
A solution of phosphate-buffered formaldehyde has been found to preserve cell morphology as well as surface and intracellular antigenicity with minuscule cell aggregation and cell loss. Only fixed cells will stand the effects of detergent treatment.
Controls:
Evidence for specificity of the cytokine staining should be based on parallel studies of isotype controls, staining with the secondary antibodies alone and an abolishment of immunoreactivity by preabsorption of the cytokine-specific antibody with the corresponding cytokine protein.
Stimulation of PBMNC for cytokine production:
The strongest and most diversified cytokine production is seen after PMA-ionomycin activation of Ficoll separated PBMNC. Both monocytes and lymphocytes will be activated. The cells are co-cultivated with PMA (1 ng/mL), ionomycin (500 nM) and harvested after 4 hours. After 4 hours, one can expect to see IL-1a, IL-1ß, IL-1ra, IL-2, IL-3, for various periods. If cells are harvested after 3 hours, one can find monokines such as IL-1a. However, cytokines produced by lymphocytes, IL-12 or substantial IL-10 production is not found in the cultures.
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