Intracellular Staining for Cytokines
Cells in the body produce chemical mediators called cytokines. In the past, methods used to measure the amounts of these chemicals involved isolating cells from human blood and culturing them in a medium which contains the nutrients they need to survive. The cells were then stimulated to produce cytokines and the released protein was measured in the fluid surrounding the cells. This method is not entirely satisfactory because the amounts of cytokines released are relatively small and so are hard to measure when diluted by the surrounding culture medium. It is now possible to stop the release of cytokines from cells in culture leading to build up within the cell. The cell membrane can then be permeablised allowing the use of specific monoclonal antibodies with fluorescent markers to label the cytokines trapped within the cell. The amount of fluorescent marker can be measured by a process called flow cytometry.
If you are a scientist and have access to the facilities and wish to try this procedure a protocol is outlined below. If you are not interested in the technical jargon (trust me it is dull) please follow this link to continue your journey through the node gel.
- Culture chosen cells at 37°C in 5% CO2 at 1x106 cells/ml in RPMI 1640 with 10% FCS and 1% pen/strep for 5h with stimulation e.g. PMA (10ng/ml) and iononmycin (1uM). Use 6 well plates and add 6ml/well.
- During culture brefeldin A (10mg/ml) is used to inhibit cytokine production at the level of transport through the rough endoplasmic reticulum.
- Following activation/culture period cells are harvested by gentle aspiration with a bulb pipette.
- Cells are washed twice in RPMI then resuspended in 1% BSA in PBS at approx 2x107 cells/ml.
- 50ml aliquots of cells are added to 15ml centrifuge tubes and 100ml of paraformaldehyde solution (Leucoperm solution A cat no. BUF09 Serotec) is added.
- Cells are then incubated for 15min at room temperature.
- 5ml 1% BSA in PBS is added to each tube and the samples are spun down at 300 x g for 5 minutes.
- Supernatants are removed and 90ml of saponin solution (Leucoperm solution B cat no. BUF09 Serotec) plus 10ml of the appropriate directly conjugated antibody or conjugated irrelevant isotype control are added.
- Samples are gently resuspended and incubated for 30 min at room temperature.
- Cells are washed in 1% BSA in PBS as desccibed in step 7.
- Remove cells and resuspend in cell fix (Becton Dickinson) for analysis within 24hr.
-For cell suspensions prepared by enzyme digestion or through a cell strainer 50mM mercaptoethanol can be added to the RPMI.
- Leukocyte Activation Cocktail, with GolgiPlug cat no. 550583 from Pharmingen can be used in the activation cultures. It contains PMA, iononmycin and Brefeldin A at a predetermined concentration and can be added directly to the culture wells.
-Staining for extracellular antigens should be performed berore step 5. Cells should be incubated with Fc-block (10mg/ml) before being stained with the required antibodies.
-At step 5 2-4% paraformaldehyde solution in PBS can be substituted for the leucoperm.
-To test specificity of antibodies for use in intracellular staining, it is useful to preincubate permeablised cells with excess unconjugated antibody of the same isotype as the one to be used for staining.
-If further intracellular staining steps are to be performed after step 8 then saponin must be added to the washing solution between staining steps. This is to maintain permeability of the cell because the effects of saponin are reversable.
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