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Tissue samples and clogging

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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue May 13, 2014 12:03 am

Tissue samples and clogging

I'm curious how many of you are successfully running tissue samples/non-hematopoietic cells on the CyTOF? I''m running into major issues with clogging, despite passing the samples multiple times through 35micron filters immediately prior to acquisition. Surprisingly, the most challenging samples I've encountered so far have been mouse heart samples, which don't generally cause any issues on our regular flow cytometers or sorters.

Enriching tissue samples with a Percoll gradient helps with this issue, but some of our users have expressed concerns about the selective loss of certain cell populations with these gradient enrichments, and other are interested in analyzing stromal cell populations.

Anyone have any suggestions?
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Tue May 13, 2014 2:57 am

Re: Tissue samples and clogging

Hi Adeeb,
We find that running 5x10^5 cells per sample really helps with clogging.
What concentration is your sample?
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ErinSimonds

Master

Posts: 50

Joined: Tue May 13, 2014 8:04 pm

Post Wed May 14, 2014 5:29 am

Re: Tissue samples and clogging

Adeeb, do you tend to see the clogs later in the run, or right as soon as you start? If you see them later, the clogs are probably due to cell aggregates forming in the sample loop. Cleaning the loop very thoroughly between samples with a 1:20 dilution of Citranox followed by several volumes of water will help to prevent this. As Ofir suggested, a more dilute sample will probably also help avoid clogging, perhaps by the same mechanism (i.e. preventing aggregates in the loop).

I have had good experiences running dissociated solid tumor samples (human glioblastoma), even without any gradient purification. Other folks at Stanford have run mouse skeletal muscle and human ovarian carcinomas. As you suggested in your post, testing the dissociation protocol by fluorescent flow can be very helpful, and filtering through nylon mesh is essential. Gradient purification does indeed run the risk of biasing the cell frequencies -- it can be useful, but it must be tested carefully.
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Wed May 14, 2014 2:15 pm

Re: Tissue samples and clogging

Thanks Ofir,

Unfortunately running the samples at a lower sample concentration doesn't seem to resolve the problem (though running them too concentrated certainly does make things worse).

I usually dilute these "sticky" samples to less than 2 X 10^5/ml but still encounter clogging issues.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed May 14, 2014 3:00 pm

Re: Tissue samples and clogging

HI Adeeb,

One reason the samples might be OK on flow but not on CyTOF is the fact that your samples are in MilliQ water for CyTOF rather than PBS/FACS buffer. Some people have reported having some issues with increased stickiness in MilliQ.

Unfortunately, there aren't many options.
1. Continue MilliQ washes and resuspension, and deal with stickiness by running Wash solution after every 2 samples or so. This has worked for some people doing whole blood, for instance.
2. Do all your washes in PBS, then do just the resuspension in MilliQ. This was the original CyTOF workflow, several years ago. This may help with your stickiness, as some buffer salts will remain. Unless you have a barium or other heavy-metal contamination in your buffer, the buffer salts won't make it through the quadrupole mass filter and therefore won't be killing your detector because of their high concentration.
-HOWEVER: the buffer salt *will* build up on the cones rather quickly. This won't harm the cones, per se, but *will* cause your Current peak to shift to higher Current over the course of even one day. I've seen the peak optimum/tip move by as much as 2 Current units (unsure whether it's A, or mA...) over one day of runs. So, if you do this, I would recommend re-checking the tuning halfway through your run. Also, the buffer salts will build up on the spray chamber as well: I have attached a picture of this. So, if you don't mind cleaning the cones every morning, this might be something worth trying (you should already be cleaning the spray chamber every day). Note: buffer salts are white: cell debris and most associated heavy metal debris is yellowish/brownish.


Mike
Attachments
dirty spray chamber-buffer salts.jpg
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nicole

Participant

Posts: 3

Joined: Mon Dec 02, 2013 6:42 pm

Post Wed May 14, 2014 3:55 pm

Re: Tissue samples and clogging

Hi Adeeb,

We have had issues with sample stickiness in the past as well. For us, this has been due to the following -
1. Poor fixation
2. High levels of debris/poor starting viability
3. Remaining salts from your buffers after water washes. I see that Mike has said the opposite but in our experience we see the salt residue building up in the nebulizer and blocking the sample over time (and as Mike pointed out you will need to do more frequent cleaning)

Generally if a user is having this issue, I recommend that they make their fixation buffer fresh for each experiment, check viability and carefully aspirate all their buffers off the cell pellet. You can check your fixation immediately after your fix step by looking at the scatter on flow. I also check sample quality (debris) this way once in water with the threshold set very low. We also have some users that have implemented a second fix step with 4%PFA immediately prior to their final PBS and water washes. For samples with high amount of debris or low viability I dilute samples to 0.25M/ml final (including debris and dead cells in the count). I also wash after each loop injection with wash solution and water to remove any build up. If you are using the auto-sampler, make sure you are washing in between each well and try decreasing your well volume to as low as 250ul.

-Nicole
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu May 15, 2014 3:08 pm

Re: Tissue samples and clogging

Hi all,

1. Fixation is definitely important. Most CyTOF labs recommend diluting PFA from 16% stock each time, rather than keeping a 2% stock. Also, using a 16% stock that is no more than 1 month old: even if kept covered, PFA starts to go off somewhere around 1 month. And remember, the osmotic stress of MIlliQ water is vastly different than the stress of PBS/FACS buffer: just because a particular stock of PFA is sufficiently fixing your cells for flow is NOT a guarantee that it is still sufficiently active to fix your cells adequately for CyTOF.

The characteristic behavior is that you see a nice cell pellet through all of your PBS and FACS buffer steps, and then the pellet starts to change and/or disappear once you start your MilliQ water washes.

2. Erin: can you comment on the citranox vs Wash solution efficiency, in terms of running stickier samples? Is Wash just not sufficient?
We've never done the citranox: partly b/c we almost always run just PBMC/WB, and partly b/c of my chromatography background being against introducing detergents/"soapiness" to any sort of fluidics system (and the bubbles that can form).


Mike

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