Hi Dina,

when we stained for live/ dead (L/D) stains I usually used V506 from Ebioscience (which is now also available in Thermo Fisher's catalog).

http://www.thermofisher.com/order/catalog/product/65-0866-14

The peculiar issue with this dye is that only works in BSA free PBS, but not in FACS buffer (PBS + 0.5% BSA). I learned that mistake to my dismay. (The V506 is an aminoreactive dye binding free amine groups, BSA interferes with that binding) Therefore, I would suggest that you stain with the above mentioned L/D stain in a small volume of BSA free PBS and top-up with a bigger volume BSA containing PBS prior to your acquisition to bind the access L/D stain. I hope that helps with the overstaining issue.

All the best & good luck with your experiment!

Michael

Thanks for your answer Michael, especially for the BSA part. I actually have serum in my medium so I guess I must look for L/D stain that is not affected by serum.

Hi Dina,

Michael is absolutely right with his hint that proteins as BSA interfere with live/dead cell staining. Moreover: usual Phospho-staining protocols base upon very short incubation times after adding the stimulus (as you mentioned already) and harsh fixations with MetOH or commersial buffers like Phosflow - both not allowing a post-stimulus L/D cell stainig.

To circumvent these issues, I perform L/D stainig BEFORE stimulus (in protein free PBS) to define cells that are already dead (due to freezing). After washing, I rest the cells for a while in complete medium @37°C (to be ´non-stressed´) before adding the appropiate stimulus to induce Phosphorylation of the protein of choice. Than perform phosphostain according to protocoll.

Consequently, initially L/D+ stained cells can be excluded in your PhosphoFACS analysis (plus: use stringent scatter gating!). However, with this strategy (and I don´t know a better option) you can´t exclude cells dying upon the short stimulus phase (though quite rare in frequencies in my hands).

Since there a several L/D dyes available (and I don´t know if which one you´ll use) : you should pretest the stability of your dye upon your Fixation method. Thaw one PBMC aliquot, perform L/D cell stainig, keep 1/2 in FACS buffer and perform with the other 1/2 exact fixation/permeabilization as you´ll use for your Phospho stainig. Comparing both samples will allow for evaluating the stability of your L/D dye (frequencies and MFI) - thus ensuring that you´ll detect dead cells despite the harsh fixation.

Good luck

Cheers,

Sylvia

Hello Sylvia,

This is brilliant, thanks.

I have one more question, I can see from your protocol that you do not serum-starve your cells prior to stimulation. I am wondering if you detect any background signalling in your unstimulated controls?

Thanks again.

Dina.

Dear Dina,

indeed - I compared STAT Phosphorylation after TLR Ligand-mediated IL-6 secretion (thus declaring w/o TLRL as negative sample [background] and including a high positive control [exogenous IL-6]). In these comparisons it was not necessary to starve the cells for reducing background. However, I would guess that the protocoll above should also work with a starving ´slot´ after L/D staining (which is in my hand stable for a couple of hours).

Good luck

Cheers

Sylvia

These are very helpful answers. Thanks so much.