Saturday, October 18, 2003

Description
A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular cytokines at single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens, as in the regular staining protocol, then fixed and permeabilized to allow for anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in freshly-prepared cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-gamma, TNF-alpha, IL-2, and IL-4, a combination of PMA (a PKC activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies are used. To induce IL-6, IL-10 or TNF-alpha production by monocytes, stimulation of PBMC's with LPS is used.

Procedure
Prepare target cells of interest

Stain cell-surface antigen following the Surface Staining Protocol. The choice of the surface marker depends on the experimental question.

After the last wash, fix the cells by adding 100 祃 of Fixation Solution while vortexing the tube and incubate in the dark at room temperature for 20 minutes.
Add 1 ml of Permeabilization Buffer to each tube, centrifuge for 5 minutes and aspirate supernatant.
Repeat step 4.
Resuspend cells in 100 祃 of Permeabilization Buffer and incubate in the dark at room temperature for 5 minutes.
Dilute the optimal concentration of fluorochrome-conjugated anti-cytokine mAb in 20 祃 Permeabilization Buffer and add to the appropriate tube. Mix by vortexing. A good range of conjugated antibody for this application is 0.5-0.06 礸/10(6) cells. eBioscience fluorochrome-conjugated anti-cytokine antibodies are also available in 20 祃/test sizes. If using these reagents, add 20 祃 of pre-titrated antibody to the appropriate tube.
Incubate in the dark at room temperature for 20 minutes.
Add 1 ml of Permeabilization Buffer, centrifuge for 5 minutes and aspirate supernatant.
Resuspend the cell pellet in 0.5 ml of Flow Staining Buffer.
Run on a flow cytometer and analyze.


Recipes


Supplies


Tips