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Cisplatin quench

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marthabrainard1

Contributor

Posts: 20

Joined: Wed Aug 25, 2021 2:10 pm

Post Thu Jan 27, 2022 6:22 pm

Cisplatin quench

Hi CyTOFers,

We are trying to switch up our protocol and with the changes, it makes the most sense for us to perform viability staining in a plate. However, we can't quench cisplatin at 5x or 10x this way. Has anyone used another reagent to quench cisplatin or stained in deep well plates? Would 1-2mls be enough to quench the reaction? We currently quench at 5 or 10x with CSB.

Happy Thursday,
Martha
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mleipold

Guru

Posts: 4324

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jan 27, 2022 6:32 pm

Re: Cisplatin quench

Hi Martha,

What staining volume are you using? And how deep are your wells (shallow well ~300uL?)?

I regularly use deepwell plates for my stainings. I use these 1mL deepwell round bottom Polystyrene plates: https://www.fishersci.com/shop/products ... ord=278605

I know some people don't like using polystyrene, but I've never had any obvious issue with cells sticking, and the fact that the wells are transparent (rather than milky-translucent) means that I can visually confirm my pellets after every step.


Mike
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marthabrainard1

Contributor

Posts: 20

Joined: Wed Aug 25, 2021 2:10 pm

Post Thu Jan 27, 2022 7:29 pm

Re: Cisplatin quench

Hi Mike, we normally perform the cisplatin dilution in 15mL conicals with 125uL of 2x cisplatin added to 125 uL of CD45 stain, we then quench in 5 mLs of CSB. We want to lower the volumes of cisplatin to be able to stain in a shallow well plate, but weren't sure if we could lower the quench volume.
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jobagins

Participant

Posts: 13

Joined: Thu Oct 22, 2020 6:45 pm

Post Thu Jan 27, 2022 8:18 pm

Re: Cisplatin quench

Hi MB

You can stain cispt in regular plate - staining volume 100uL, (so you can do 50 cis 50 cd45) and quench with 100uL of CSB with increased BSA concentration (10%) for this wash only. What quenches the Pt is actually bsa so its enough to increase % without increasing volume. Follow with one regular CSB wash
How long you do cisPt? I recommend 5min RT 5uM final concentration, then I dont know if 5min is enough for cd45

Let me know your thoughts

JoB

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