Storage after staining with anti-fluorochrome Abs

[nuirat]

Hello ,

I'm quite new in the Cytof world, just came over from flow and currently run my first experiments.

I stain primary human cells in a panel of 24 mass cytometry Abs, 2 of which are anti-fluorochromes (FITC, APC) and 1 is anti-Biotin.
As of the literature, we determined that we can probably store the cells for 1 weeks in the fridge w/o loosing the signal of the regular surface staining. Freezing is no option as we envision intracellular staining as well and the freezing process does not seem beneficial for this one...
However, in Flowcytometry, I would always (maybe I'm too picky but I worked with rare populations) acquire PFA-fixed samples (stored in the fridge and additionally covered from light) within 3 days of staining not to loose any signal. So, I am wondering now, whether when I keep my samples for a week in the fridge before acquisition on Helios, the fluorochrome would faint and the connection to the secondary (Cytof) Ab would potentially (partially) get lost? Or does the fixation for Cytof create a stable link between the 2 Abs?

I was wondering if someone has already experience in this issue/tried it out, so that I can abandon the 7-day storage right away or try it out w/o a too high risk.

Thanks a lot in advance,
nuirat

[mleipold]

Hi Nuirat,

I can't directly address the question of secondary stain stability over several days.

However, I can say, when I do secondary stains with anti-fluorophore antibodies, I don't worry about covering them from light. I recently did a project where I used both anti-PE and anti-APC, and didn't cover the plates during primary, secondary, live dead, fix (though that was in the fridge overnight, so dark), perm, Iridium, or during acquisition.

So, as far as I know, the anti-fluorophore antibodies don't seem to be terribly sensitive to any photobleaching effects on binding.


Mike