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Aggregates and cell loss in bone marrow samples

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mayanlevy

Participant

Posts: 9

Joined: Thu May 11, 2017 10:26 am

Post Fri Oct 16, 2020 5:23 am

Aggregates and cell loss in bone marrow samples

Hi,
We're having a bit of a problem while trying to find progenitor populations in bone marrow.
The protocol for CyTOF includes staining the cells, fixation, methanol perm for 15min, intracellular staining, fix and washes in 800g and Ir staining.
After washing the methanol, we had 5 million cells. However after Ir staining we were left with 3 million. Meaning we lost 2 million cells in the process. In addition, our CyTOF keeps clogging because of strange aggregates keeping us with insufficient cells to find rare populations.
This feels a bit excessive and I was wondering where in the process we had it wrong.
Methanol is ice cold (-20C), added drop-wise, centrifuges were done in 800g post fix and 600g pre-fix.
Is it possible the cells were more vulnerable after the methanol and exploded in such speeds?
Thanks
Mayan
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komal2000

Participant

Posts: 16

Joined: Mon Aug 29, 2016 12:55 pm

Location: University of Helsinki, Finland

Post Fri Oct 16, 2020 7:36 am

Re: Aggregates and cell loss in bone marrow samples

Hi, This is very common in phosphoflow experiments, after fix and perm you loose lots of cells. Where do u stain the cells I mean are you using tubes or eppendorf. I usually do the staining in eppendorf to avoid cell loss.
I had the similar issue with the clogging before but at the end of the protocol filtering very slowly with 40micron helped a lot and also depends on the type of cells you are staining. Before running the samples in CyTOF take a look at the cells in the microscope if you still see clumps.
Some of the rare populations are very sensitive to fix and perm buffers for instance stem and progenitor cells. Lucky you are still have 3 million cells for running CyTOF.
I would suggest you to do a pretest and optimize the centrifugation steps to find better G for your cells (make sure your centrifugation step should not effect signaling proteins and viability).


Sr
Komal
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taxkourel

Participant

Posts: 14

Joined: Sun Sep 03, 2017 3:46 pm

Post Fri Oct 16, 2020 10:48 am

Re: Aggregates and cell loss in bone marrow samples

Are you using fresh or cryopreserved cells? If the later, what is their viability after thawing? U can try 300g for your prefix speed i have seen that make a difference.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Oct 16, 2020 1:15 pm

Re: Aggregates and cell loss in bone marrow samples

600g before fixation seems very high. We typically don't do more than 480g, and sometimes less.

Also, you don't say how many cells you're starting out with (ie, plating from the beginning), so it's hard to comment on whether you're disproportionately losing cells late in the protocol.

What density are you running your cells at?
In my experience, bone marrow often needs to be run slower (more dilute) than cryo PBMC. I'd start at 0.6M/ml (~180-200 events/sec?) and see whether that helps your clogging problem.

Where are the clogs happening? At the nebulizer? In the sample capillary (grounding nut to nebulizer)? In the PSI (assuming you have a Helios)?

What is the expected frequency of your progenitor cells (based on flow, etc)?

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