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How to prevent "sticky" cells clogging machine

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xiaoxiaoUC

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Joined: Sun Feb 04, 2018 4:48 pm

Post Sun Feb 04, 2018 10:22 pm

How to prevent "sticky" cells clogging machine

Hi guys, I am using women placental membrane cells for CyTOF experiment. I started to meet machine clog issue recently. CyTOF core technician said the machine was running well with beads but once my sample was on, the clog formed, so he had to filter my cells many times. I think it may be caused due to the cell components of placental membrane: big size of decidua stromal cells with mucus.... I always washed the cells many times, but I don't know it's enough to wash out the mucus.


Do you have some protocol or method can prevent "sticky" cells forming aggregation?

Is there any other reason can cause this issue?

Much appreciate it!

Xiao
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MCOlivier

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Posts: 47

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Mon Feb 05, 2018 8:48 am

Re: How to prevent "sticky" cells clogging machine

Hi Xiao,

You can try 2 mM EDTA to reduce 2+ ions dependent adhesion... It works well on hepatocytes.
I know also protocols for sorting of musclucar stromal vascular fraction in which you wash cells 7 times because of "stickiness" of the digested tissue.

Wish you chance on it !

Olivier
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xiaoxiaoUC

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Joined: Sun Feb 04, 2018 4:48 pm

Post Mon Feb 05, 2018 2:54 pm

Re: How to prevent "sticky" cells clogging machine

Thanks so much, Olivier! Your advice is very helpful.

Do you know which step I can add EDTA? Before staining or after staining? Will EDTA influence antibody binding?

Thanks again!

Xiao
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mleipold

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Location: Stanford HIMC, CA, USA

Post Mon Feb 05, 2018 3:41 pm

Re: How to prevent "sticky" cells clogging machine

Hi Xiao,

I usually have 2mM EDTA in my staining buffer (PBS+EDTA+BSA), and at least with my panel, it doesn't affect binding of my antibodies. However, there are some antibodies whose target epitopes are 3D structures that contain metal ions, so you'll just have to test it.


Mike
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xiaoxiaoUC

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Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Mon Feb 05, 2018 4:45 pm

Re: How to prevent "sticky" cells clogging machine

Much appreciate it, Mike!!! I will try!

Xiao
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xiaoxiaoUC

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Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Tue Feb 06, 2018 2:41 am

Re: How to prevent "sticky" cells clogging machine

Mike

One more question: Do you know what can cause iridium signal variation among samples and experiments?

Thank you so much!

Xiao
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RDevine

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Post Tue Feb 06, 2018 3:12 am

Re: How to prevent "sticky" cells clogging machine

Hi Xiao,

We've been experimenting with an EDTA and DNAse solution but we haven't really perfected it. We designed this to be added in at any time, as soon as clumping is noticeable but when we set up an experiment to test out a titration of EDTA and DNAse solution cells that were reliably clumping stop clumping all of a sudden. We've experimented with 10U/ml DNase and 5 mM EDTA solution thats added to the sample and left in 37C incubator for 20 minutes after the Ir incubation. We've also tried just EDTA and we've had some success but we haven't really been able to experiment with it thoroughly.

Ray
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xiaoxiaoUC

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Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Tue Feb 06, 2018 3:39 am

Re: How to prevent "sticky" cells clogging machine

Ray,

That's a good point. Our samples are frozen samples, so there were dead cells and cell debris in each vial. I can imagine the free DNA also can cause the "sticky" environment. I will try your method, and let you know the result.

Thank you!

Xiao
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Feb 06, 2018 4:28 am

Re: How to prevent "sticky" cells clogging machine

RDevine wrote:Hi Xiao,

We've been experimenting with an EDTA and DNAse solution but we haven't really perfected it. We designed this to be added in at any time, as soon as clumping is noticeable but when we set up an experiment to test out a titration of EDTA and DNAse solution cells that were reliably clumping stop clumping all of a sudden. We've experimented with 10U/ml DNase and 5 mM EDTA solution thats added to the sample and left in 37C incubator for 20 minutes after the Ir incubation. We've also tried just EDTA and we've had some success but we haven't really been able to experiment with it thoroughly.

Ray


One point to note here is that EDTA and DNase generally do not play well together; DNAse is a calcium-dependent enzyme, so adding EDTA is probably inadvertently inhibiting the activity of your DNAse.

EDTA and DNase prevent clumping for very different reasons - EDTA inhibits calcium-dependent cell activation and adhesion, while DNase degrades sticky DNA that's released from dying cells and binding up the rest of your cells. You're best off figuring out which one is causing issue in your specific prep and then targeting that.

Also, while it can help to add these to help deal with clumps that form during staining, keep in mind that your cells are already fixed by the time they get to the instrument so activation induced adhesion should not really be a problem any more, and neither should cell death.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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xiaoxiaoUC

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Post Tue Feb 06, 2018 2:02 pm

Re: How to prevent "sticky" cells clogging machine

Adeeb


Thanks for your notes.

Xiao
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