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Low viability with cryopreserved bone marrow samples

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taxkourel

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Posts: 14

Joined: Sun Sep 03, 2017 3:46 pm

Post Thu Sep 14, 2017 11:05 pm

Low viability with cryopreserved bone marrow samples

Hello!

I am a just getting started with CyTOF and got stuck already.

I am using cryopreserved human bone marrow samples from patients but I am having poor viability especially after an overnight rest step.
I tried this with PBMCs (no overnight rest though , this is when I was just testing things out) and viability after surface staining was consistently above 80% and usually in the high 80s/low 90s.

I start with ~10 million cells total
I thaw as follows (gentle pipetting all the way):
1) Thaw cryovial quickly (in bath or in my hand)
2) add 1ml of warm benzonase RPMI (20% FBS) slowly and then dump into Falcon with 9 ml warm benz//se media
3) spin down 200G 15mins X1-At this point I have >90% viable cells
4) spin down again 200GX15 min-Viability drops to 70%
5) rest overnight in 20% FBS (no benzonase) (concentration ~3-5 million/ml) in 6 well plates
6) viability next morning is 50%!!! :-(

I could stain with just the surface panel right after I thaw but I 'd like to include a cytokine/intracellular stain so I need to rest them.
On top of that after I use the benzonase free medium I get some clumping and loose a few cells at this step too. So in the end I start with 10 million cells and end up with 2 and lots of dead cells/debris. :-(

I get similar results irrespective of bone marrow infiltration my malignant cells (in case cancel cells are dying easier). Samples have all been stored within the last 2 yrs.
If you have any ideas on how to improve this at any step of the way please let me know.

Many thanks!
Tax
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dtdavani

Participant

Posts: 12

Joined: Fri Sep 11, 2015 9:52 pm

Post Mon Sep 18, 2017 2:44 pm

Re: Low viability with cryopreserved bone marrow samples

Hi Tax,

We are working with frozen BM samples on a regular basis in the context of Phospho-signaling and/or Cytokins. I think the following could help to increase the viability.
1- We thaw and wash the samples with RPMI complete(10%FCS)+benzo to avoid clumping. Spins are 400 g 8 min
2- Unless the overnight is mandatory for your cytokines, you could try shorter resting. We rest the cells for 2 hr 37' C CO2 incubator for all of our experiment. 2 hr has shown reduction of base line signaling or Cytokine production ( IFng, IL2, IL17, IL4, TNFa) reasonably for us and reduces cell death.

Ultimately you could compere the above method with your SOP side by side.

I hope this helps,

Dariush
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taxkourel

Participant

Posts: 14

Joined: Sun Sep 03, 2017 3:46 pm

Post Mon Sep 18, 2017 3:10 pm

Re: Low viability with cryopreserved bone marrow samples

Thank you Dariush!
I do loose quite a few cells with the overnight rest so might just do 2hrs as you suggest!
Thank you for posting!

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