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Metals-Minus-? and cytof gating

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ChantelMcS

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Posts: 7

Joined: Thu Sep 04, 2014 6:07 pm

Post Thu Sep 04, 2014 8:44 pm

Metals-Minus-? and cytof gating

Cytof gating is perplexing me. In flow, I’d make gates based on population breaks and if that was not clear/obvious, I’d make an FMO. With my cytof data, I have 3 different B-cell markers that are not clear. Does it make sense (to save on ab’s etc) to make one control that would be the same as my panel minus the 3 challenging abs or must I make a “metals minus one”? Based on DVS’s chart, the 3 troubling markers do not contribute to each other.
One more question: When making “metals-minus” kind of controls, do we need to include ab-metal (that based on DVS cross talk chart) cannot contribute to the signal in question?
Thank you
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maecker

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Joined: Thu Oct 31, 2013 7:58 pm

Post Fri Sep 05, 2014 8:40 pm

Re: Metals-Minus-? and cytof gating

I would certainly favor a "MMM" (metal minus multiple) rather than "MMO" (metal minus one) approach, as long as there is no significant cross-talk between the channels being left out. And, as you suggest, it should be fine to leave out other antibodies as well, as long as they don't contribute to signal in those channels, and as long as they're not needed for gating of populations relevant to those markers. -Holden Maecker
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Sep 05, 2014 8:57 pm

Re: Metals-Minus-? and cytof gating

I would agree with Holden that "MMM" is probably faster. However, if that doesn't help, then going back and doing the individual MMO's would be the next thing.

Could you show us some of your data in question? Ideally, starting from Ungated down through your gating strategy.

Underneath the Reply window, there is a tab called Options, and a tab called Upload attachment....you can use that to show us some data plots.


Mike
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ChantelMcS

Participant

Posts: 7

Joined: Thu Sep 04, 2014 6:07 pm

Post Mon Sep 08, 2014 3:54 pm

Re: Metals-Minus-? and cytof gating

Thank you for the comments. I have attached (via screen shots) the gating and the MM-3 along with 4 samples. They were acquired on different days however normalization beads were used. Samples had my complete panel and the MM-3 had only ab's that were needed to gate on population of interest (Live B-cells as defined by CD45+3-20+) and any metals that could contribute to signal in Dy162, Nd144, & Sm149. (samples: thawed human PBMC)
Attachments
MM-3 and 4 samples.png
Gating .png
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ChantelMcS

Participant

Posts: 7

Joined: Thu Sep 04, 2014 6:07 pm

Post Mon Sep 08, 2014 7:38 pm

Re: Metals-Minus-? and cytof gating

and in Flowjo X ....
Attachments
Screen shot gating in X.png
gating
Screen shot CD20 in x.png
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Sep 09, 2014 8:42 pm

Re: Metals-Minus-? and cytof gating

Cells and Beads.pdf
(115.51 KiB) Downloaded 384 times


HI Chantel,

I want to make sure I'm understanding your gating:

In the first picture, for file 15328-127.fcs, you start with a plot of Ce140 vs Eu153 for Ungated cells. This gave you a gate you called "beads". You then gated "beads" with Ir191 channel.

Do I understand this correctly?


If so, gating initially for 2 bead channels for Ungated would only give you Beads and Bead-Cell doublets.....not Cells. Therefore, your Ir191 gate would be for cells that had stuck to beads.

I would recommend going back and trying Ir191 vs Ce140. Ir+Ce- would be cells, Ir+Ce+ would be bead-cell doublets, and Ir-Ce+ would be beads. I have attached an example.

Cells could then be further refined by Ir191 vs Ir193.

-Mike

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