Flow cytometry can be used to analyze various intracellular molecules including phosphorylated signaling proteins and cytokines. Cytokines, small signaling molecules secreted by many cell types, can be detected by flow cytometry in activated cells with the aid of secretion inhibitors, such as monensin or brefeldin A. These compounds prevent the export of newly synthesized proteins by disrupting the ER-Golgi transport machinery. For experimental treatments with stimulation periods of up to 4-6 hours, the secretion inhibitor can be present during the entire incubation period. If the stimulation period is longer than 4-6 hours, the secretion inhibitor should be added for only the last two hours of the incubation.
There are many variables that must be optimized for individual flow cytometry experiments such as antibody incubation time and temperature. Furthermore, to stain intracellular molecules, the cells need to be fixed in suspension and then permeabilized before the detection antibody is added. This fixation/permeabilization treatment allows the antibody to pass through the plasma membrane into the cell interior, while keeping the morphological characteristics used to sort the cells intact. Alcohols, such as methanol or ethanol are commonly used to permeabilize the cell. Cold methanol is typically used as a permeabilization agent when using flow cytometry to detect phosphorylated proteins and transcription factors, because it can increase the reactivity of antibodies to certain nuclear antigens. The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for the staining of intracellular molecules for flow cytometric analysis.
Note: When surface and intracellular staining are to be performed in the same sample, it is advisable that the surface staining be performed first because fixation/permeabilization treatments might decrease the availability of surface antigens.
Please read the protocol in its entirety before starting.
Note: Staining of surface antigens may be done at this point.
Note: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in PBS (or HBSS), starting with the concentration suggested in the product datasheet. Incubate for 20-30 minutes in the dark and wash as in step 3.
Note: For a negative control, a separate set of cells should be stained with an isotype control antibody.
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|Detection of Phospho-ERK1/ERK2 by Flow Cytometry. The Jurkat T cell line was unstimulated (light orange; open histogram) or treated with 50 μg/mL PMA for 10 minutes (dark orange; filled histogram). Cells were incubated with the Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Monoclonal Antibody (Catalog # MAB1018) or an isotype control antibody (Catalog # AB105C; blue; open histogram), followed by staining with a Fluorescein-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0112). The cells were fixed in paraformaldehyde and permeabilized with ice cold methanol.|
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