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MaxPar 159Tb antibody signal observed in 160/161

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hwtchen

Participant

Posts: 9

Joined: Thu May 07, 2015 6:35 pm

Location: Sickkids, Toronto, Canada

Post Thu Jun 25, 2015 5:17 pm

MaxPar 159Tb antibody signal observed in 160/161

Recently we started to acquire all the MaxPar available mass channels when titrating a newly MaxPar conjugated antibody. While titrating an antibody (GFP) tagged with 159Tb we observed unexpected signals in channel 160 and dimmer in 161 but not in 158 (see plots, link below). Further digging showed another previously conjugated antibody with the same issue. We have obviously ruled out isotopic impurity but are not certain if it is due to abundance sensitivity since it is only observed in +1 not -1 channel. Could this be due to contamination of the MaxPar 159Tb metal stock? Has any one else observed this with their reagent? Any input is welcomed. Thanks.
https://drive.google.com/open?id=0B9p4t ... E5qWUlvck0
Tina Chen
CyTOF Research Project Coordinator, Guidos Lab
CyTOF Operator, Sickkids- UHN Flow Cytometry Facility
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jun 25, 2015 5:42 pm

Re: MaxPar 159Tb antibody signal observed in 160/161

Hi Tina,

Were you using any other markers in your experiment panel, or just DNA and 159Tb?


Mike
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hwtchen

Participant

Posts: 9

Joined: Thu May 07, 2015 6:35 pm

Location: Sickkids, Toronto, Canada

Post Fri Jun 26, 2015 2:25 pm

Re: MaxPar 159Tb antibody signal observed in 160/161

Also Cis-Pt, that is all.
Tina Chen
CyTOF Research Project Coordinator, Guidos Lab
CyTOF Operator, Sickkids- UHN Flow Cytometry Facility
<<

mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jun 26, 2015 3:11 pm

Re: MaxPar 159Tb antibody signal observed in 160/161

Hi Tina,

Try taking some of your 159Tb stock and diluting it ~1e5-1e6, and running it in solution mode like you would tuning solution. You should then be able to to see whether you still see 160-161.

You can also try staining BD Kappa capture beads with your antibodies of concern, and running them like cells.


Doing both would help you figure out whether the metal stock is contaminated, or your antibody stock, or whether it's coming from some other source (Ir, Pt, buffers, etc).


You can also buy trace-metal basis 99.9% pure TbCl3 from Sigma: #212903. You can make up a 50mM stock of that in L buffer, and use it for comparison....or as a substitute for the stock from Fluidigm.

Mike

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