Phospho-flow: orthovanadate

Today's post is a little side note of something I found when trying to optimize the dish soap protocol. I couldn't get the protocol working well for detection of phosphoryated epitopes (phospho-flow), so I was testing phosphatase inhibitors in an attempt to block dephosphorylation that I thought was occurring post-fix. Anyway, it turns out that this is probably useful for a different reason.

If you include sodium orthovanadate prior to fixation (during the stimulation or for about 30min of resting), it can preserve the low-level phosphorylation events that are normally continually erased during homeostasis. For example, Tregs are known to exert immunosuppression via continual consumption of endogenous IL-2 through the high affinity IL-2 receptor, CD25. In the absence of stimulation with exogenous IL-2, Tregs exhibit minor pSTAT5 signal when stained overnight. This signal increases to represent nearly all Tregs when sodium orthovanadate is included in the cell culture prior to fixation. This finding may be useful for anyone wishing to monitor homeostatic phosphorylation states.

Mouse splenic CD4 T cells stimulated for 20min at 37C

This makes a lot of sense with what we know about the biology. There is always going to be a little IL-2 around, and Tregs (and probably ILCs, activated T cells too) will grab this via CD25. That keeps the equilibrium. But we should see the impact of that IL-2 binding to CD25 as a phospho-STAT event, and we know that homeostatic IL-2 is important because removing CD25 on Tregs is not good for Tregs (see here, here and here). So, we should be seeing something, and the fact that we aren't suggest a failure of sensitivity or a failure to retain the signal.

Preparing a solution of pervanadate would probably be even more effective, but this is a bit more work. Some other phosphatase inhibitors that I tried were fluorescent, so be careful and check on unstained cells, especially if you try cocktail tablets.

Reagents:

Sodium orthovanadate (see manufacturer instructions for preparation)

Foxp3 eFluor 450

pSTAT5 PE-eFluor 610

Phospho staining buffer: TBS + 2.5% FCS + 2mM EDTA

Basic protocol:

Pre-stain for viability (other stable fluorophores may be stained at this point)

Stimulate at 37C in a TC incubator

Directly add 4% formaldehyde (10% formalin) to the wells

Incubate 30min at RT

Spin down, resuspend in -20C methanol

Incubate 30min at 4C (or on ice)

Wash 2x with phospho staining buffer

Block 15min with 2% rat + mouse serum

Stain overnight in phospho staining buffer

Wash 2x with phospho staining buffer

Black-bellied Hummingbird, Costa Rica