1. Culture adherent cells to 80 to 90% confluence in complete medium.
2. Serum-starve the cells, when needed, with serum free medium for 12-16 hours prior to stimulation (serum starvation lowers basal phosphorylation levels).
3. The serum starved cells are either left unstimulated or stimulated with an approrpriate activator while the cells are still attached.

Note:

1. It is important (at least for some surface receptor based activations) not to trypsinize to detach the cells, since trypsin may cleave ligand binding extracellular domains of receptors. If the cells have to be detached, we recommend to detach the cells using protease free cell detaching solutions.
2. Cells can be detached first, for non-receptor mediated activation, using trypsin. Some receptors are resistant to trypsin treatment, so it has to be determined empirically if a given cell line can be detached before conducting a desired activation.
3. Stimulation conditions vary. Researchers should optimize for their own stimulation conditions.
4. At the end of cell treatment, detach the cells as quickly as possible using 1X trypsin solution.

As soon as the cells are fully detached, disperse the cells into single cell suspension by pipetting, and fix the cells immediately by adding an equal volume of Phosflow Fix Buffer I (cat. no. 557870) pre-warmed to 37°C. Incubate at 37°C for 10 min.

Note:

1. It is important to minimize the time between the end of cell stimulation and cell fixation to preserve phosphorylated targets.

• Spin down the cells and remove the supernatant.
• Optional: Wash the cells once with 1X PBS.

Permeabilize the cells by adding cold Phosflow Perm Buffer III (cat. no. 558050) and incubating on ice for 30 min.

Note:

1. At this stage, cells can be permeabilized with either Phosflow Perm/Wash Buffer I, Phosflow Perm Buffer II, or Phosflow Perm Buffer III, but we find that Phosflow Perm Buffer III works the best for all our current phospho-specific antibodies.
2. Cells can be stored at -20°C at this stage for later staining.

• Spin down the cells and wash the cells twice with the Staining Buffer (1X PBS, 1% FBS, and 0.09% sodium azide).
• Resuspend the cells in the Staining Buffer at cell density of 5 to 10 million cells per ml, and use 100 µl per test for staining.