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.
The following materials will be needed:
- Trypsin/EDTA solution (Cat. no. 25300-054)
- Complete Cell Culture Medium (e.g., DMEM plus 10% FBS)
- PBS (Invitrogen Cat. nos.11885-076 and 26140-079)
- Cell Extraction Buffer (Cat. no. FNN0011)
- Orbital Plate Shaker (Lab Line Titer Plate Shaker Cat. no. 4625-EA)
- p38 MAPK [pTpY180/182] phosphoELISA (Cat. no. KHO0061)
- p38 MAPK Total phosphoELISA (Cat. no. KHO0071)
- Nunc MicroWell 96-Well Microplates (Cat. no. 167008)
- T75 cell culture flask (Cat. No. 156499)
- 15mL Conical Tube (Cat. No. 339651)
Cell extraction buffer formulation:
- 10 mM Tris, pH 7.4 • 2 mM Na3VO4
- 100 mM NaCl • 1% Triton X-100
- 1 mM EDTA • 10% glycerol
- 1 mM EGTA • 0.1% SDS
- 1 mM NaF • 0.5% deoxycholate
- 20 mM Na4P2O7 • 1 mM PMSF (stock 0.3 M in DMSO)
Prepare Protease Inhibitor Cocktail (Sigma Cat. no. P-2714) according to the manufacturer’s guideline as a 10x stock. Add 100 μl per 1 ml Cell Extraction Buffer.
Please see Figure 1 for a general overview of the procedure.
- Grow cells to desired level of confluency in a T75 flask.
- Decant or aspirate the medium.
- Add 2–3 ml fresh warm trypsin/EDTA solution. Transfer the flask to a 37°C incubator.
- Wash with warm PBS. Aspirate.
- After 5 minutes, tap the side of the flask, and examine the flask under a microscope for lifting. If necessary, return the cells to the incubator for an additional 5–10 minutes, with occasional tapping, until lifting is complete.
- Quickly quench the Trypsin reaction by adding 5–6 ml Complete Cell Culture Medium.
- Transfer the cells to sterile 15 ml conical tubes.
- Pellet the cells by centrifugation at 300 x g for 7 minutes.
- Decant the supernatant.
- Wash the cells by pipetting 10 ml medium into each conical tube and resuspending the pellet. Collect the cells by centrifugation at 300 x g for 7 minutes.
- Resuspend the washed cells in complete cell culture medium.
- Enumerate cell density. For most applications, the cell density should be adjusted to 5–25 x 104 cells/ml cell culture medium. It is important to note that this value may require some optimization for each specific application. Cell doubling time is an important factor to be considered when adjusting cell density at the beginning of an experiment.
- Plate 200 µL of cell culture (i.e., 10,000–50,000 cells) into the wells of the sterile 96-well cell culture plate. Incubate the cells for 18 hours at 37°C.
- Stimulate the cells as desired. In the example presented below, HeLa cells were treated with 100 μM anisomycin for 60 minutes at 37°C.
- At the end of the treatment, aspirate the media from the wells.
- Wash the cells by pipetting 200 µL ice-cold PBS into each well. Remove the PBS by aspiration and repeat two times for a total of three washings.
- Aspirate the medium. Pipette 25 µL protease-inhibitor-supplemented Cell Extraction Buffer into each well. Incubate the plate on ice for 30 minutes.
- Thoroughly mix the contents of each well by pipetting up and down 5–6 times. A multi-channel pipette is desirable for this application. This procedure causes the cells to lyse. At this point in the procedure, the extracts are ready for analysis. Alternatively, the extracts may be stored in the filter bottom plate at –20°C for future analysis. Frozen plates should be thawed on ice in preparation of completing the assays.
- Place the plate on an orbital shaker and mix for 1 minute.
- Prepare the phosphoELISA kits. Sample Wells: Pipette 95 µL Standard Diluent Buffer (included in the kits) into the wells of the phosphoELISA plates designated for samples. Transfer 5 µL cell extract from the filter plate into the sample wells of the plates. Place the plates on an orbital shaker to thoroughly mix the contents of the wells. Standard Wells: Prepare standards as indicated in the assay protocol and pipette into designated wells.
- Complete the phosphoELISA as directed by the assay protocol.
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.
Figure 2. The data shows the result of normalizing data obtained with p38 MAPK [pTpY180/182] phosphoELISA (Cat. no. KHO0061) to data obtained with the p38 MAPK Total phosphoELISA (Cat. no. KHO0071). Anisomycin treatment increases the level of phosphorylation of p38 MAPK at threonine 180 and tyrosine 182. This data also shows that the quantity of p38 MAPK [pTpY180/182] normalized to total p38 MAPK is directly proportional to the number of cells seeded in the wells of the filter bottom plate.