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CD66b lost in Frozen vs Fresh vs PBMCs

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TomerWeizmann

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Posts: 22

Joined: Mon Apr 07, 2014 11:58 am

Location: Weizmann Institute of Science - Israel

Post Thu Jun 13, 2019 6:09 pm

CD66b lost in Frozen vs Fresh vs PBMCs

Dear CyTOFers,

We would appreciate your insights on loss of CD66b+ populations in Frozen vs Fresh Human PBMCs prep after Ficoll.
The cells were frozen in FBS+10%DMSO.

We are familiar with the literature on the issue addressing MDSCs and their sensetivity to freezing, e.g.:
'Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determined by multiparametric single cell mass cytometry analysis'
https://bmcimmunol.biomedcentral.com/ar ... LaWaItadxU

viSNE plot attached.

Any thoughts on how to preserve the cells as so not to lose them?

Many thanks!
Attachments
CD66b.PNG
Dr. Tomer Meir Salame
Flow Cytometry Unit
Life Sciences Core Facilities
Weizmann Institute of Science
E-mail: tomer-meir.salame@weizmann.ac.il
http://wws.weizmann.ac.il/Biological_Se ... etry-about
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eganio

Participant

Posts: 15

Joined: Wed Nov 07, 2018 8:00 pm

Post Thu Jun 13, 2019 6:27 pm

Re: CD66b lost in Frozen vs Fresh vs PBMCs

Hi Tomer,

Since preparation of PBMCs usually depletes granulocytes (which express CD66), I would imagine that you're simply losing a very small population of grans that remained in the PBMC layer after Ficoll prep. Preservation of this small subset might prove very difficult.

A couple questions:

1. What is your PBMC freezing protocol?
1. Are you freezing PBMCs in liquid nitrogen?
2. What is your recovery protocol from frozen?

-Ed
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TomerWeizmann

Contributor

Posts: 22

Joined: Mon Apr 07, 2014 11:58 am

Location: Weizmann Institute of Science - Israel

Post Fri Jun 14, 2019 9:55 am

Re: CD66b lost in Frozen vs Fresh vs PBMCs

Hi Ed,

Thank you for the reply.
Both are after Ficoll (and from the same sample). The only difference was the freezing.

Answers:

1. After the isolation of the PBMCs we washed the cells with 3 ml of Maxpar Cell Staining Buffer and centrifuged the cells at 400 G for 10' at RT (acceleration ,9; brake,5). Then, we removed the supernatant, resuspended the cells in residual volume and add 1 ml of ACK and incubate for 2'. Next, we washed the cells with 5 ml of Maxpar Cell Staining Buffer and centrifuged the cells at 400 G for 10' at RT (acceleration ,9; brake,5). Following this, we removed the supernatant, resuspended the cells in 3 ml of Maxpar Cell Staining Buffer and count the cells with using Trypan Blue exclusion. Finally, we centrifuged the cells at 400 G for 10' at RT (acceleration ,9; brake,5), removed the supernatant, resuspended the cells in residual volume and added 1,5 ml of the CryoStor cell cryopreservation media CS10.

2. We put the cells in the Mr. Frosty overnight and the following day we performed the thawing of the samples and subsequent mass cytometry staining.

3. We thawed the vial directly on the bench, washed the cells with 5 ml of Maxpar Cell Staining Buffer and proceeded with the protocol.
(we are now trying a more gentle recovery protocol from the frozen PBMCs and increased the yield of live cells from 50-60% to 80- 90%. We included the use of a water bath to thaw the frozen vials and gently adding dropwise the PBMCs on top of 10 ml of RPMI supplemented with FBS 10% to wash the cells.)


Many thanks,
Tomer
Dr. Tomer Meir Salame
Flow Cytometry Unit
Life Sciences Core Facilities
Weizmann Institute of Science
E-mail: tomer-meir.salame@weizmann.ac.il
http://wws.weizmann.ac.il/Biological_Se ... etry-about
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eganio

Participant

Posts: 15

Joined: Wed Nov 07, 2018 8:00 pm

Post Fri Jun 14, 2019 2:50 pm

Re: CD66b lost in Frozen vs Fresh vs PBMCs

Hi Tomer,

A gentler recovery of the frozen cells would probably help, although you will still be losing a portion of an already small subset. Also, you might want to try slowly introducing the DMSO into the freezing medium instead of using the Cryostor. Our protocol involves re-suspending the cells in 1mL 100% FBS, then slowly (drop-wise) adding 1mL 20% DMSO/FBS with gentle swirling to gradually incorporate the DMSO and create 2mL 10% DMSO/FBS. However, I'm not sure this will show a significant difference in cell viability compared to the Cryostor.

Are you specifically interested in granulocytes? If so, I would suggest that instead of PBMC prep, you should employ a simple RBC lysis in a hypotonic buffer, which will recover a very large number of grans. After running the samples through CyTOF, you can gate out the residual RBCs and platelets using CD235ab and CD61 antibodies on the same channel, which is what we typically do. You can then use the RBC/platelet-excluded .fcs files for your viSNE plots.

-Ed
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TomerWeizmann

Contributor

Posts: 22

Joined: Mon Apr 07, 2014 11:58 am

Location: Weizmann Institute of Science - Israel

Post Fri Jun 14, 2019 3:16 pm

Re: CD66b lost in Frozen vs Fresh vs PBMCs

Dear Ed,
Thank you for the informative reply. I agree. We are following similar suggestions.

Our main concern is losing MDSCs (Myeloid-derived suppressor cells, which are also CD66b+) and not the Granulocytes.

Much appreciated.
Tomer
Dr. Tomer Meir Salame
Flow Cytometry Unit
Life Sciences Core Facilities
Weizmann Institute of Science
E-mail: tomer-meir.salame@weizmann.ac.il
http://wws.weizmann.ac.il/Biological_Se ... etry-about
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jun 14, 2019 3:32 pm

Re: CD66b lost in Frozen vs Fresh vs PBMCs

Hi Tomer,

I'm not that familiar with MDSCs. In your fresh samples, what is their frequency (frac of LiveIntactSinglets(LIS))? Do you find this to be replicable (ie, multiple draws from the same donor give similar frequencies)?

The reason I ask: because of how tSNE works, I can't tell how large that population is as a fraction of total cells. Therefore, it's hard for us to determine whether freezing is the *only* issue, rather than freezing + isolation. For example, you can see basophils in PBMCs, but at least some of comparisons of fresh vs frozen (or PBMC vs WB) data I've seen shows that the basophil Freq changes depending on the isolation. Part of that is the low freq of basophils, and part of that is they don't all isolate efficiently on Ficoll.

So, if MDSCs are, say, reproducibly 5% of total LIS in the fresh PBMCs and reproducibly 0.1% in the frozen PBMCs, then yes I'd say that freezing is the culprit since that's the only real variable. But if you have a range of percentages of MDSCs in the fresh over multiple draws from the same donor, then isolation efficiency is a compounding variable.


Mike
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TomerWeizmann

Contributor

Posts: 22

Joined: Mon Apr 07, 2014 11:58 am

Location: Weizmann Institute of Science - Israel

Post Sun Jun 16, 2019 8:58 am

Re: CD66b lost in Frozen vs Fresh vs PBMCs

Hi Mike,

Thank you for the input.
Live singlets in fresh was 80-90% and in thawed 50-60%.
It was replicable as well as other varialbes.
I agree that the freeze-thaw proccess is the cause.

Thank you all again,
Tomer
Dr. Tomer Meir Salame
Flow Cytometry Unit
Life Sciences Core Facilities
Weizmann Institute of Science
E-mail: tomer-meir.salame@weizmann.ac.il
http://wws.weizmann.ac.il/Biological_Se ... etry-about

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