Flow cytometric analysis of live mouse splenocytes, untreated (left column) or treated with Cell Stimulation Cocktail (with Protein Transport Inhibitors) (500X; right column), using IFN-γ (XMG1.2) Rat mAb (APC Conjugate) #32793 (top row) or concentration-matched Rat Isotype Control (APC Conjugate) (bottom row), and co-stained with CD3 (17A2) Rat mAb (FITC Conjugate) #86603.
1. Prepare the cell suspension in the appropriate cell culture medium.
2. To obtain a 1X final concentration, add the Cell Stimulation Cocktail (with Protein Transport Inhibitors) (500X) directly to the cell suspension at 2 µL/mL.
3. Incubate the cells at 37°C in an atmosphere that contains CO2.
4. Harvest the cells for the immunoassay of interest.
Note: The concentration of cells and incubation time will vary depending on the desired results. This product is an ethanol solution and susceptible to evaporation. Keep tightly sealed when not in use.
Store at -20ºC tightly sealed to prevent evaporation and protected from light. This product is stable for at least 6 months when stored at -20ºC.
Cell Stimulation Cocktail (with Protein Transport Inhibitors) (500X) is an ethanol solution that contains the phorbol ester, PMA (phorbol-12-myristate-13-acetate), the calcium ionophore, Ionomycin, and the protein transport inhibitors Brefeldin A and Monensin. When added in vitro to cell suspensions, the PMA and Ionomycin activate the cells, leading to increased cytokine production. The Brefeldin A and Monensin limit protein secretion, allowing for intracellular detection of the accumulated cytokines by downstream immunoassays.
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