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How to prevent "sticky" cells clogging machine

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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Feb 06, 2018 3:57 pm

Re: How to prevent "sticky" cells clogging machine

Hi all,

As our standard treatment, the HIMC adds Benzonase to our cell media (RPMI + pen/strep/glutamine + heat-inactivated FBS) during thawing. We then do an extra benzonase-media wash after the first centrifugation; both benzonase+media washes are to help remove DNA present from cells lysed during freeze-thaw. After that, the cells in my experiments are almost always in CyFACS (PBS + 0.1% BSA + 2mM EDTA).

See here for our standard live cell surface phenotyping protocol: viewtopic.php?f=11&t=258

In my experience, yes, samples with lower viability are more likely to clump, mostly in the CyFACS *after* the 2 benzonase+media washes. So, EDTA doesn't always take care of the problem.

However, I will also state: clumping can also be a donor-specific occurrence. For example, I was recently working on a study with multiple PBMC timepoints (~6 week period) for each donor. For one donor, 0 of the 7 timepoints clumped. For another donor, 6 of the 7 timepoints clumped as soon as the cells "saw" CyFACS (ie, were taken out of benzonase+media, and before they "saw" antibodies), and continued clumping through the entire experiment. There was no significant difference in viability or recovery (>85% viability, >70% recovery) between the two donors, and the likelihood is very small that only one donor would have been mis-processed or mis-stored multiple times over the course of ~6 weeks. The time series for each (healthy) donor was the same as well, so it's unlikely that it was a reaction to the study's vaccination (also, one of the affected donor's clumping timepoints was Day 0 pre-vaccination).


So, unfortunately, sometimes these things just happen, and we haven't figured out a way to *stop* or *reverse* clumping once it starts.


Mike
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue Feb 06, 2018 4:21 pm

Re: How to prevent "sticky" cells clogging machine

Hi Xiao,

I would echo what has been said already in the other great comments.

The most important point is to perform benzonase/DNase treatment while the cells are thawing and resting. There are almost always dead cells in thawed cryopreserved samples and these dead cells will almost always release DNA that will form a sticky goo that will clump up the live and dead cells. It is critical to have benzonase/DNase around to break this up. In addition to the media components that Mike mentioned, we typically also include heparin, but I'm honestly not certain that this is absolutely needed.

However, just like Mike's experience, we have also had cells clump after this step, and we can't find any clear rhyme or reason to this. In fact, on at least two occasions, we have intentionally used cells that we thought would clump to test different "declumping" solutions and even cells that had previous created a lot of clumps wouldn't consistently clump again in spite of processing them in exactly the same way.

Finally, I would just suggest that once you start getting clumps that you thoroughly clean out the fluidics, as we tend to find that cell clumps in the fluidics tend to stimulate the creation of more cell clumps (even from cells that wouldn't otherwise clump).

best of luck,

Greg
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xiaoxiaoUC

Contributor

Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Thu Feb 08, 2018 10:58 pm

Re: How to prevent "sticky" cells clogging machine

Mike and Greg

You guys suggestions are very helpful. I tried Mike's protocol using DNase, but I incubated my cells for 15 min in 37c water bath. I also removed cell debris. There were a lot cell debris in my sample. I think they were generated during the tissue processing. Following Greg said, I looked at my sample after filtering and before putting it on the machine, and I still can see the cells were easily to form linear cloud not really like clump. I think women placenta tissue has so many mucus unlike PBMCs. That's why it made my CyTOF life very tough...So I pipetted up and down at least 10 times before putting the cell tube onto the machine. This time, I do get an improvement by these modifications. The machine was running much smoother than before.

Thank you all!!

Xiao
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avanderzwan

Participant

Posts: 2

Joined: Mon May 15, 2017 10:13 am

Post Fri Feb 09, 2018 8:44 am

Re: How to prevent "sticky" cells clogging machine

Hi Xiao,

I also work with placental cells and as you mentioned it might come from tissue processing. In the past I used Collagenase/DNase to isolate my cells from the tissue. It would make the tissue quite 'slimy'' during filtering. Now I have switched to Accutase and the whole process is a lot less 'slimy'.
I am sure you do this, but filtering before putting the placental cells on the CyTOF also gets rid of a lot of clumps.

Good luck!

Best,
Anita
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xiaoxiaoUC

Contributor

Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Wed Feb 14, 2018 8:51 pm

Re: How to prevent "sticky" cells clogging machine

Anita

Thanks for your suggestion! Could you give me the Cat # of your Accutase. I want to try it as the same as yours. And when did you put it? Just during the membrane processing?

Did you use placenta membrane cells to run CyTOF? do you have experience that even the running speed is slow as "100~150" events/sec, there was still "double events" in the data set? I think this was caused by the cell stickiness.

Sorry about so many questions. Much appreciate it!

Xiao
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avanderzwan

Participant

Posts: 2

Joined: Mon May 15, 2017 10:13 am

Post Sat Feb 17, 2018 11:24 am

Re: How to prevent "sticky" cells clogging machine

Hi Xiao,

I use Accutase from Gibco Ref#A11105-01. I mince the tissue, then add the Accutase, use the GentleMacs and then put the tissue in a shaking water bath for 1hour.

I run both basalis and parietalis on the CyTOF. In my experience the parietalis looks a lot cleaner on the CyTOF than the basalis. The basalis I really have to run at 100-150events/sec, but the parietalis can be run higher. I see more 'double events' in my data set for basalis than for parietalis.
Do you isolate only the parietalis, or do you also include the chorion? The chorion is indeed very sticky, from the start of isolation.

Hope this helps,

Anita
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