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Long CyTOF runs in Helios

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mayanlevy

Participant

Posts: 4

Joined: Thu May 11, 2017 10:26 am

Post Thu Dec 19, 2019 7:07 am

Long CyTOF runs in Helios

Hello all :)
Did not see any recent (=Helios relevant) post on the matter.

I want to run barcoded samples (unsure yet about the volume. Could be between 6 to 13mL) but I feel like doing this continuosly might cause a significant signal reduction.

How do you do this normally? Do you stop every couple of hours to wash with water or deal with the signal reduction later?

If it's of any relevance, the samples are whole blood, diluted to 0.5 million cells per mL.

Thanks,
Mayan
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Verhoeff

Participant

Posts: 16

Joined: Wed Mar 02, 2016 10:25 am

Post Thu Dec 19, 2019 9:41 am

Re: Long CyTOF runs in Helios

Hi Mayan,

Our preferred method is to split the pooled sample into aliquots that can be run on the CyTOF over the course of the day in 45min-1hour runs when diluted. We split this after the first wash with CSB after overnight incubation with DNA intercalation. Then all aliquots are washed with the second CSB wash, but only the first aliquot is washed further with 2x H20. Others are left pelleted in a small volume. This way the amount of iridium signal is consistent across the sample run at 9:00 and the one at the end of the day.

In your case this would be ~0.75mln cells, to be diluted in 1500uL H20 (with 10% beads of course). In between the runs, while washing the next aliquot with H2O, we wash with Wash solution and H20. Every 2-3 runs we retune the CyTOF. Quick protocol first, but if it fails then Full Tuning. If the sample would be closer to 13mL it might not fit in 1 day. When that happens we only wash a portion of the pooled sample, the rest to be measured the following day is left in Ir intercalation solution. There will be a slight shift in 193Ir and 191Ir peaks, so pre-analysis gating may need to be done on each day separately if doublets are hard to distinguish.

Hope that helps,
Jan

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