Has anybody used Zombie aqua dye (fixable viability dye for FACS) and can give me an idea about the gating of the cells?

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Sudipta Tripathi

I've not used zombie aqua, but zombie red. My feeling is ambivalent. I never saw two distinct populations of live and dead, which could be for various reasons. I only saw a shift, a big shift, and considered the negative and low zombie red to be alive. My experiment was for ICCS staining, which is very different from yours. But the bottom-line is, the negative is population is the live population.

I'm assuming you used the manufacturer's protocol and using a protein free environment. Also assuming that you stain your cells before you heat kill. It can also be because your sample is heterogenous. How does your unstained sample look like?

Hope this helps.

Tabasum Sidiq

Hi Sudipa,

Thanks for your kind response. Please find attached the dot plots for unstained sample and the samples with different zombie aqua dilutions. I used the company protocol and protein-free environment (1XPBS) and incubated the samples with zombie at RT for 30 min. Is that ok?I stained the cells with zombie after heating at 65 degrees for 1 min.

Thanks,

Tabasum

Hi Martin,

Thanks for the elaborate explanation. I really appreciate. I will follow your suggestion and hope I get what I want.

Thanks

As Martin has correctly pointed out zombie dyes stain only the surface protein in the live cells. It appears that you are using a higher concentration of dye and that's probably the reason why you see 3 populations. To me it seems, the lower concentration is working better. And also the 3rd population keeps disappearing with the lowering the dye concentration. titrate your dyes every time you use a new one. it is the easiest and best way to ensure that you get an adequate signal and minimal background.

Sepideh Parsi

Hi I am wondering if anyone used this dye before fixation and IF? its signal is quite nice for IF but not sure how to do meseaurement. Actually I am getting not homogenous varied shapes of green signal in my confocal imaging some extremely strong some weak ...