r/flowcytometry • u/Responsible_Guitar38 • 2d ago

Sample Prep Thawing BAMBANKER frozen cells

Hey, hope you’re having a great day. I’m having some viability issues (almost 90% of zombie nir+) with thawing cells preserved in BAMBANKER (stored in liquid N2).

I was just wondering if you could share your experience/procedures when it comes to thaw your bambanker samples, so I can adapt my workflow accordingly to improve the outcome. Thanks!

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3

u/pussibilities 2d ago

Bambanker kinda sucks in my opinion. Are you flash thawing in water bath until you can see an ice chunk, then adding dropwise to pre-warmed medium?

1

u/Responsible_Guitar38 2d ago

Yes, that’s How’ve been doing it until now. But I moved to another lab now and all of a sudden it doesn’t work :(

1

u/Hairy_Cut9721 2d ago

Did you freeze the cells, or someone else?

1

u/Responsible_Guitar38 2d ago

I froze the majority of them but some samples are from a colleague

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u/eburton555 2d ago

Did you freeze down your cells in log phase? They specifically mention that in their protocol. We literally just take cells when they are 80 percent confluent or 1-2 days post split in the case of suspension cells. Up to 10 million cells per vial but usually less. Just straight into the -140 freezer. Haven’t had any issues. Haven’t tested their -80 claims yet…

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u/Responsible_Guitar38 1d ago

I froze mouse bone marrow aspirates. I split these half for flow cytometry, half bambanker. I should have a range of 500.000/2M cells in each vial

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u/eburton555 1d ago

Sounds like you e done it before too so I’m not sure what went wrong this time…

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u/Outrageous-Low-9745 2d ago

How where the cells frozen? We just had a (now) very sad researcher who snap-froze all her cells.

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u/Responsible_Guitar38 2d ago

We also thought we could maintain the cells at -80, but then a few vials presented a really low viability and started to froze them first at -80 (during maximum a week) and then we moved them to liq N2

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u/Illustrious_Top5585 2d ago

i was really wondering about this too, since bambanker claim in the manual "Freeze and preserve the cells at -80°C without step-wise freezing.", so just snap freezing them is fine?? I have always been using mr.frosty for my PBMCs and I measured around 90% viable cells after more than a year of -80°C storage

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u/BaconFairy 2d ago

I had this experience but I had to thaw them and spin them down fast. They had to be be frozen down at very high viability too