The procedure presented below describes a facile method for studying signal transduction events with adherent cells (HeLa, MCF-7, BALB/c 3T3, etc.). In this procedure, cells are gently lifted from their culture vessel, placed into wells of a 96-well filter bottom plate, and stimulated as desired. At the end of stimulation, cell culture medium is removed from the bottom of the wells by gentle aspiration using a vacuum manifold. The cells are then washed with PBS, aspirated, and lysed within the wells by addition of Cell Extraction Buffer. The cell extracts are then assayed using Invitrogen p38MAPK [pTpY180/182] and p38MAPK (total) phosphoELISA kits.
Add Protease Inhibitor Cocktail (Cat. No. 78429) directly to the lysis buffer or extract to produce a 1X final working solution (10 µL of Halt Protease Inhibitor Cocktail per mL of cell lysis buffer).
Figure 2 shows the results obtained with p38 MAPK [pTpY180/182] phosphoELISA (Cat. No. KHO0071). The data presented show that anisomycin treatment increases the level of phosphorylation of p38 MAPK at threonine 180 and tyrosine 182, and that this is directly proportional to the number of cells seeded.
HeLa cells were seeded into the wells of the plate at densities of 3,000, 6,000, and 12,000 cells in 200 μL cell culture media. The seeded cells were incubated for 18 hours at 37°C, then treated with anisomycin (100 μM) for 60 minutes, or left untreated. At the end of the incubation period, the cells were lysed by the method described above, and assayed with the p38 MAPK [pTpY180/182] phosphoELISA kit.
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