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Cell counter

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MayaBr

Participant

Posts: 3

Joined: Wed Nov 11, 2020 8:32 am

Post Wed Jun 23, 2021 9:11 am

Cell counter

Hi!

I'm looking for recommendations on most efficient way to count cells before antibody titration and staining.

I work at an SRL center, where we perform the Ab conjugation, titration, staining and cyTOF acquisition as a service. We currently count cells with Countess II life technologies (https://assets.thermofisher.com/TFS-Ass ... ter_UG.pdf). We suspect that machine over estimates the true number of cells.

Thanks!

Maya
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jun 25, 2021 6:31 pm

Re: Cell counter

Hi Maya,

In my experience, different cell counters give slightly different numbers for the same sample. For example, using the default settings on our Vicell vs TC20, the TC20 seems to give slightly higher cell counts as a general rule. This seems to come down to how each of the counters counts: the Vicell has circularity and another couple parameters in addition to lower and upper cell diameter cutoffs, whereas the TC20 seems to mainly (just?) have cell diameter cutoffs.

In terms of experimental design, it has been our opinion that *consistency* (precision/reproducibility) for initial plating is more important than absolute accuracy of cell count (assuming there's maybe a 10% difference or less between different methods). Put differently, pick one cell counter method and stick with it for a given application.


So, we use the Vicell when thawing and plating large numbers of samples, since it's something we can set up and walk away from. And then we use the TC20 for the sample-by-sample dilution immediately before running on the CyTOF.


Mike
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TomerWeizmann

Contributor

Posts: 43

Joined: Mon Apr 07, 2014 11:58 am

Location: Weizmann Institute of Science - Israel

Post Sat Jun 26, 2021 9:16 am

Re: Cell counter

Hi Maya,
I agree with Mike.
In our SRL we also have good experience with the DeNovix CellDrop, and you can tweak its settings to achieve accurate counts.
Good luck,
Tomer
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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JamesW

Contributor

Posts: 37

Joined: Tue Nov 21, 2017 6:59 am

Post Thu Jul 01, 2021 8:38 am

Re: Cell counter

Dear Maya,

Recently we did some demos of a few different cell counters. We tested;

Countess III FL: Cheapest fluorescent machine. We found it was an outlier in the cell counts as its autofocus seemed to struggle, you can manually focus but it was a bit fiddly.
Luna-II: Brightfield only, seems ok but we didn't feel it outperformed the TC20.
Biorad TC20. brightfield only. We have had this for a while, its quick and easy but I think struggles with lower quality samples. Also its does take a picture but the screen is so small its hard to double check things.
Luna-FL: Nice screen, manual focus but easy to do. Is an aggressive shade of orange.
Luna-Fx: Quite nice features but also expensive.
Denovix CellDrop: No slides so potentially a lower running cost and nice features but again pretty expensive day 1.

Mostly they gave similar counts on good quality samples but if the sample is very complex or messy PI/AO on the florescent machines outperformed Trypan blue on brightfield if you want viability.

Probably we will get a Luna-FL for accurate counting in the culture room and keep our TC20 for a quick check before running on the Helios. I agree with Mike that consistency may be more critical than absolute accuracy.

One other conclusion was that if you have the time a microscope and haemocytometer is still a solid option.
James
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desireeBCRT

Contributor

Posts: 23

Joined: Mon Jan 06, 2014 11:56 am

Post Thu Jul 01, 2021 12:36 pm

Re: Cell counter

Hi Maya,
we use a TC20 for counting each sample before running on the CyTOF. I like the option to export the image to a USB key, which also give you the opportunity to see an enlarged image on your computer screen. If samples are very clumpy, I sometimes send users the image to stress the point that they need to improve sample quality.
Desiree

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