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SuperSampler cleaning during run

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mayanlevy

Participant

Posts: 2

Joined: Thu May 11, 2017 10:26 am

Post Mon Jul 03, 2017 7:09 am

SuperSampler cleaning during run

Hi,

I am preparing for a big run in the CYTOF, using both barcoding and the SuperSampler. But using the Nolan Normalizer we can see that the signal goes down with time. I was wondering maybe it'll be beneficial to clean the CYTOF briefly every 30 minutes or so just to clean some of the debris and prevent possible clogging.

Also wondering if this becomes an issue after more than 2-3 hours of run with the SuperSampler?

Thanks :)
Mayan
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CRStevens

Contributor

Posts: 26

Joined: Thu Jul 17, 2014 5:07 pm

Post Tue Jul 11, 2017 5:18 pm

Re: SuperSampler cleaning during run

Hi Mayan,

There is always inherent drift in signal with the technology, but are you suggesting that there is more drift with the SuperSampler vs say the PSI unit or injection loop?

I've used both methods and haven't observed this, but haven't really done a side-by-side comparison, either.

We have run larger barcoded studies where the samples have been run for 8+ hours and we haven't seen large enough drops in signal that we were unable to normalize or analyze the data. It is also important to have as clean a sample as possible to make sure the drift is minimized. If there is residual PBS from your washes this can make your drift much more exaggerated.

-Chad
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GregBehbehani

Master

Posts: 78

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue Jul 11, 2017 7:46 pm

Re: SuperSampler cleaning during run

Hi Mayan,

I would agree with Chad completely.

If the drift you're referring to is the decrease in the bead intensity over time, that is an expected characteristic of ICP-MS. The beads were created specifically to allow for the normalization of this signal decrease.

If you're referring to signal loss over time that is greater than than the decrease in the bead signal, that is caused by prolonged exposure of your cells to pure water (due to the loss of antigens and/or antibodies from the cells). The best way to deal with this is to minimize the time the cells are in pure water (wash them out of intercalator into pure water in aliquots every few hours). Keeping your sample cold also helps slow this process down.

The only reason to clean the super-sampler during a run would be if you think it's dirty (i.e. you're getting clogs or seeing cell debris).

best of luck,

Greg
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AdeebR

Grand master

Posts: 167

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Jul 11, 2017 10:41 pm

Re: SuperSampler cleaning during run

Hi Mayan,

While I agree with Chad and Greg that you can certainly normalize for drifts in in signal based on the beads, it's important to appreciate that decreasing signal intensity does likely mean a lower signal to noise ratio, particularly for low intensity cell signals, and that normalization ultimately can't compensate for increased noise in the data.

As a result, when doing long barcoded acquistions we typically keep our bulk sample in PBS and prepare ~1-2M cell aliquots in H20, which we acquire in ~1hr increments.

After each acquisition, we run a QC check on the data and if the EQ bead signal intensity has dropped below a defined level (e.g., 20% of starting value, which usually takes ~4-5hrs of acquisition time), we pause acquisition and retune to reoptimize DV. Since we typically switch back to loops to retune, we simultaneously run a clean cycle on the SS.

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

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