96-Well Sample Preparation for Suspension Cells

The procedure presented below describes a facile method for studying signal transduction events with suspension cells (Jurkat, Raji, THP-1, etc.). In this procedure, cells are plated into the wells of 96-well filter bottom plates and stimulated as desired. At the end of the 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.

Materials:

• Sterile 96-well filter bottom plates, 1.2 μm pore size (Millipore Cat. no. MSBVS1210)
• Vacuum manifold (Whatman Cat. no. 7705-0102)
• PBS, ice-cold (Cat. no. 70013-032)
• Cell Extraction Buffer (Cat. no. FNN0001)
• 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)

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)

Protease Inhibitor Cocktail (Sigma Cat. no. P-2714), prepared according to the manufacturer’s guideline as a 10x stock. Add 100 μl per 1 ml Cell Extraction Buffer.

1. Grow cells to desired level of confluency in a T75 flask.


2. Decant or aspirate the medium.


3. Add 2–3 ml fresh warm trypsin/EDTA solution. Transfer the flask to a 37°C incubator.


4. Wash with warm PBS. Aspirate.


5. 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.


6. Quickly quench the trypsin reaction by adding 5–6 ml complete cell culture medium.


7. Transfer the cells to sterile 15 ml conical tubes.


8. Pellet the cells by centrifugation at 300 x g for 7 minutes.


9. Decant the supernatant.


10. Wash the cells by pipetting 10 ml of medium into each conical tube and resuspending the pellet. Collect the cells by centrifugation at 300 x g for 7 minutes.


11. Resuspend the washed cells in complete cell culture medium.


12. Enumerate cell density. A hemacytometer is recommended. For most applications, the cell density should be adjusted to 25–100 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.


13. Plate 200 μl of cell culture (i.e., 50,000–200,000 cells) into the wells of the sterile 96-well filter-bottom plate. Incubate the cells for 24 hours at 37°C. The filter plate is designed to retain particles, while permitting the flow of liquids from the bottom of the plate.


14. Stimulate the cells as desired. In the example presented below, Jurkat cells were stimulated with 20 μg/ml anisomycin for 60 minutes at 37°C.


15. At the end of the stimulation, place the filter bottom plate on the vacuum manifold and remove cell culture medium from the bottom of wells by gentle aspiration.


16. Wash the cells by pipetting 200 μl ice-cold PBS into each well. Remove the PBS from the bottom of the wells by gentle aspiration. Repeat two times for a total of three washings.


17. Pipette 30 μl protease-inhibitor-supplemented Cell Extraction Buffer into each well. Incubate the plate on ice for 30 minutes.


18. Thoroughly mix the contents of each well by pipetting up and down 5–6 times. A multi-channel pipette is desirable for this application. 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.


19. Place the plate on an orbital shaker and mix for 1 minute.


20. 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 thorougly mix the contents of the wells.


21. Standard Wells: Prepare standards as indicated in the assay protocol and pipette into designated wells.


22. Complete the phosphoELISA™ as directed by the assay protocol.

The results presented in Figure 1 were obtained with the p38 MAPK [pTpY180/182] phosphoELISA™ (Cat. no. KHO0061). The data clearly demonstrates that anisomycin treatment increases the level of phosphorylation of p38 MAPK at threonine 180 and tyrosine 182. The data presented in Figure 1 also show that the quantity of p38 MAPK [pTpY180/182] measured with this kit is directly proportional to the number of cells seeded into the wells of the filter bottom plate. The results presented in Figure 2 were obtained with the p38 MAPK Total phosphoELISA™ (Cat. no. KHO0071). This kit was designed to permit normalization of p38 MAPK phosphorylation state. The data presented in Figure 2 show that anisomycin treatment has no impact on the amount of total p38 MAPK measured in cell lysates, indicating anisomycin increases p38 MAPK [pTpY180/182] by enhancing phosphorylation state (Figure 1), rather than altering total p38 MAPK level. The data presented also show that the quantity of total p38 MAPK measured with this kit is directly proportional to the number of cells seeded into the wells of the filter bottom plate.

Figure 1. p38 MAPK [pTpY180/182] phosphoELISA™ kit. Anisomycin treatment of Jurkat cells increases the level of p38 MAPK [pTpY180/182] phosphorylation.

Figure 2. p38 MAPK [pTpY180/182] phosphoELISA™ kit. Anisomycin treatment of Jurkat cells does not change the level of p38 MAPK (Total) expression.