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Cisplatin for cell ID

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nicole

Participant

Posts: 3

Joined: Mon Dec 02, 2013 6:42 pm

Post Wed May 13, 2015 5:05 pm

Cisplatin for cell ID

Has anyone tried using cisplatin for cell-ID in place of or at the same time as Ir191/193? I have 2 different labs attempting to use it as a means of barcoding however in both cases we have encountered what appears to be dissociation of cells. They are using natPt (Pt195) for viability, Ir to identify all cells and barcoding with Pt194, Pt198 and Rh103. The barcoded channels should always be also positive for Ir but were not always. The Ir positive cells should have always also been positive for one of the barcodes but were not. Instead we see a large number of Ir negative, Pt barcoded positive events. There also appear to be a large number (in comparison to non-barcoded samples) of events that are negative for both Pt and Ir but look like real cell events. It also appeared like there was a large number of events that were either Ir or Pt positive but negative for all of the markers. We would love to know if anyone else has seen this and has suggestions on improving the results

Thanks!

Nicole
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed May 13, 2015 7:11 pm

Re: Cisplatin for cell ID

HI Nicole,

Could you share some plots with us?

Also:
1. For the cisplatin, are all the stocks being purchased from Fluidigm?
2. Could you lead us through the protocol(s), starting with live cells (either fresh or thawed)? Particularly, at what step is the barcoding being performed?
3. What did the background look like, in *all* acquired channels?

Assuming your RBC lysis/removal has been efficient, you should really only have Ir+Ir+ events as Intact cells. If you're getting a lot of Ir- Pt+ or Ir- marker+, it makes me wonder how good your fixation was. Generally speaking, cells take up Ir well after perm, but if they're not fixed well, they'll burst during MilliQ washes. In that case, you'll have a ton of debris streaking to various levels in every channel, and low to no Ir signal.


Mike
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Wed May 13, 2015 8:02 pm

Re: Cisplatin for cell ID

Hi Nicole,

It might also be worth noting the Ir/Rh intercalators would be binding primarily to nucleic acids, whereas the cisplatin would also bind to proteins. So depending on your cell prep, I could envision that RBCs, platelets, microparticles or other non-nucleated debris might appear more positive for cisplatin than Ir.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC

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