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2014-Yao et al-J Immunol Methods

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Ofir

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Location: US, CA

Post Mon Nov 10, 2014 10:20 pm

2014-Yao et al-J Immunol Methods

"CyTOF supports efficient detection of immune cell subsets from small samples"
Yi Yao, Rebecca Liu, Min Sun Shin, Mark Trentalange, Heather Allore, Ala Nassar, Insoo Kang, Jordan S. Pober, Ruth R. Montgomery

J Immunol Methods, 2014, Nov. 4 [online]
http://dx.doi.org/10.1016/j.jim.2014.10.010
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Ofir

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Post Tue Nov 11, 2014 10:44 pm

Number of cells for CyTOF

Hi all,
I'm very interested to hear about your experience in running various numbers of cells on CyTOF and CyTOF2.
What's the smallest number you can run (mouse / human / other)?
Do you have any tips and tricks you want to share?

Thanks!
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mleipold

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Location: Stanford HIMC, CA, USA

Post Tue Nov 11, 2014 11:49 pm

Re: NUmber of cells for CyTOF

HI Ofir,

I think there are a few topics contained in that question.

1. What cell populations are you looking at, and how rare are they?
-The Yao et al paper touches on this a bit: "Indeed, our study was not designed to identify the full spectrum of cells in each subset, but rather to determine the number of input cells necessary to identify cells labeling with a particular defined set of markers."
If you're just wanting to look at total CD4+, you'll need fewer cells.

2. What are you wanting to do with the cell data?
- Are you just counting them in your collection of however many cells? For instance, antigen-specific T cells are sometimes reported as count per 10 million cells.
- Are you wanting to actually calculate medians and such? If so, for statistical purposes, you'll probably need several tens to low hundreds of cells in order to have confidence in your medians, CVs, etc.

3. A couple years ago, we ran a similar experiment on our CyTOFv1. With even a bit more experience, my cell recovery would probably be a bit higher, but this was the trend that day.
"Cell #, plated" "Live intact Singlets"
200000 28315
400000 55035
500000 75524
750000 116182
1000000 174862

In summary, the 200K gave a recovery of 14%, 500K gave 15%, while the 1mil gave a recovery of 17%. Note: this includes the cell transmission efficiency loss of 70-80%, as well as all the losses with washing and other sample processing. If I'm reading Yao et al correctly, their numbers also reflect these sources of loss.

So, we at the HIMC generally ask our customers for at least 500K/sample, plated, in order to have a decent chance to go down to some smaller subsets (even just memory T cells), while ensuring half-decent statistics.


-Mike
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Ofir

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Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Wed Nov 19, 2014 4:41 pm

Re: Number of cells for CyTOF

Thanks Mike,
Very good points about the purpose of the experiment. I am more interested in the cell transmission efficiency as a function of number of cells injected in the CyTOF. Thanks for your data!

If anyone else has data on cell transmission efficiency and cell numbers, please share it. It doesn't have to be a perfectly controlled experiment to be useful insight to the community :-)
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AdeebR

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Location: NYC

Post Mon Dec 01, 2014 10:53 pm

Re: 2014-Yao et al-J Immunol Methods

Hi Ofir,

Here's an attachment of some transmission efficiency testing that I did on our CyTOF2. The input cell number in this case is the actual number that was injected into the loop (counted and serially diluted immediately prior to injection), so not factoring in cell losses during staining/sample processing. All the tests were done using the same loop (the "better" one on our instrument).

I should also note that I had set this experiment up specifically to measure the incidence of doublets, and the doublet frequencies shown in the table are a calculation based on accurate measurement of known doublets.

-Adeeb
Attachments
CyTOF_efficiency_testing.pdf
(166.26 KiB) Downloaded 347 times
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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Ofir

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Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Mon Dec 01, 2014 11:12 pm

Number of cells for CyTOF

Thanks Adeeb!
This is very interesting. There is actually an increase in event transmission efficiency when you introduce fewer cells, AND you are getting fewer doublets:
Input - Efficiency(%) - Doublets(%)
125,000 - 45.7 - 10.74
250,000 - 43.8 - 18.15
375,000 - 41.1 - 26.37
500,000 - 39.8 - 34.5

Have you tried fewer cells still?
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Dec 01, 2014 11:40 pm

Re: 2014-Yao et al-J Immunol Methods

Hi Ofir,

I would like to point out that if you have the same volume for each sample, then the concentration is less for the 125K sample than it is for the 500K sample.

Therefore, you would expect that the doublet frequency would be lower, due to better resolution between events because of the lower concentration.


MIke
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komalkumaralienboy

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Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Tue Dec 02, 2014 7:22 am

Re: 2014-Yao et al-J Immunol Methods

Hi,

I have tested up to 4 million cells for rare populations in CyTOF 2, i believe if the epitopes are abundant example CD4 and CD8 , 0.2 to 0.5 million cells are enough for efficient detection. for rare population like stem cells i would say 1 to 2 million cells are necessary.
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TomerWeizmann

Contributor

Posts: 43

Joined: Mon Apr 07, 2014 11:58 am

Location: Weizmann Institute of Science - Israel

Post Tue Dec 02, 2014 5:26 pm

Re: 2014-Yao et al-J Immunol Methods

From our experience, generally, cell recovery is 40%, 44%, 47% for 500K, 250K, 100K, respectively.
Dr. Tomer-Meir Salame
Head, Mass Cytometry Unit
Life Sciences Core Facilities
Weizmann Institute of Science
E-mail: tomer-meir.salame@weizmann.ac.il
https://www.weizmann.ac.il/LS_CoreFacil ... etry/about
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vmotta

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Joined: Mon Jan 26, 2015 10:34 pm

Post Wed Sep 21, 2016 7:22 pm

Re: 2014-Yao et al-J Immunol Methods

Hello all,

Nice discussion and thanks for sharing your data.

I have few clients asking for the length of acquisition per sample in order to estimate costs and put in their grant application.

As you have discussed it will depend on the subpopulation of interest and how rare it is.

Mike, mentioned that he asks clients to provide around 500K cells. How long would that take to acquire? If you could share your experience on the average length of acquision per sample I would appreciate.

I have learned that analysis of rare events in traditional flow would require between 100-400 positive events in order to have 10%-5% standard deviation based on poisson distribution.

Would that apply for CyTOF if you are doing cluster analysis using SPADE or VISNE? For instance, if you have a sample stained with 30-40 markers as control and another sample stained with the same 30-40 markers plus an antigen-specific tetramer. How many positive events (antigen specific cells) would be necessary to be able to see the antigen-specific T cell cluster in one sample but not in the other?

Thanks

Vinicius
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