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Whole blood staining

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avinash1

Participant

Posts: 13

Joined: Fri Jun 16, 2017 1:59 pm

Post Tue Jan 07, 2020 7:33 pm

Whole blood staining

Hi CyTOFers!

Happy New Year 2020.

I am planning to do whole blood staining with a custom made 36 marker panel. Instead of 103Rh I plan to use 198-Cisplatin because I already have cisplatin in my lab.

Question - When should I plan to stain the cells with Cisplatin live/dead ?

A. Heparin block > Antibody cocktail staining > Lyse > Cisplatin live/dead > Fix.
B. Heparin block > Cisplatin live/dead > Antibody cocktail staining > Lyse > Fix.
C. Heparin block > Antibody cocktail + Cisplatin live/dead staining > Lyse > Fix.

Thanks :)
Avi
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue Jan 07, 2020 9:43 pm

Re: Whole blood staining

Hi Avi,

Any of these should work, but each may require slightly different cisplatin concentrations, so you'll still want to titrate your cisplatin staining for whichever approach you choose. There are two key things: First, the more total protein in your tube, the more cisplatin you will need, and you may get a slightly lower difference in signal between live and dead; Second, you must do the cisplatin before the fix (which seems to be the case in all of your proposed approaches).

I would think the most important variable is what you're planning to measure after the surface stain and if you think cells will die during your surface staining and processing. If you're staining fresh whole blood, any option should work and you may not need to do a live-dead at all; there will be almost no dead cells present in fresh whole blood, so you could just include an apoptotic marker (e.g. cleaved-PARP) and not bother with cisplatin (though a reviewer might give you a hard time about this). If you think for some reason you may have dead cells, and the dead cells will look different in your subsequent intracellular staining, then I would probably perform the cisplatin as close to the fix step as possible, so that the live-dead signal is most representative of the antigens of interest at the time of fixation (which in this case would be option "A").

best of luck,

Greg

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