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CyTOF XT

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marthabrainard

Participant

Posts: 3

Joined: Wed Oct 07, 2020 3:07 pm

Post Fri Jan 05, 2024 5:20 pm

CyTOF XT

Hi,

I was looking at some tuning data for the CyTOF XT and noticed that it is pretty different from the Helios, specifically the mean duals for 159Tb and the minimum set at 500, rather than 600,000 (or>1e6). In your opinion, what is a good parameter to track machine performance on the XT?

Thank you and happy 2024,
Martha
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ycheng

Participant

Posts: 4

Joined: Thu Jul 13, 2023 1:35 pm

Post Thu Jan 11, 2024 6:29 am

Re: CyTOF XT

Hi Martha,

We installed a CyTOF XT here and have been running it for 4-5 months. Our Tb159 mean dual at tuning is about 700-900 (sometimes is 550-650), a bit lower than Helios. This is quite similar to what other people seen on XT, too.

We usually monitor the values for the 6 channels used by EQ6 beads during tuning, as well as the median metal intensity of these 6 channels on the beads of data (using FlowJo or other similar software).

I'm also curious about what's your cell recovery rate of your XT?

Hope this helps! Thanks,

Yang
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marthabrainard

Participant

Posts: 3

Joined: Wed Oct 07, 2020 3:07 pm

Post Thu Jan 11, 2024 2:58 pm

Re: CyTOF XT

Hi Yang,

Thank you for the response. We don't always run the entire sample, sometimes we go until we reach 200,000 cells or as many cells as we can acquire per sample with the time we booked on the machine. Standard Biotools FAS told me that the recovery rate was similar to the helios at about 50-70% of cells. It's interesting to look at the values for the EQ bead channels, do you generally see a lot of variation across runs, I just took a very quick look at some of the bead data and it looks very similar to me, but this was across 4 non-barcoded samples on the same run.

Thank you,
Martha
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ycheng

Participant

Posts: 4

Joined: Thu Jul 13, 2023 1:35 pm

Post Thu Jan 11, 2024 3:43 pm

Re: CyTOF XT

Hi Martha,

We always acquire every cell for each sample unless it was a test run. However, our recovery rate wasn't so great so far, about a bit less than 50%. I usually get >80% on Helios if we do everything carefully. I feel the autosampler on XT (the carousel and the two pumps system) now has a more complicated loop system so it cause cell lost.

For Helios, we always monitor the EQ4 values. For XT, these are the values we usually have, and so far it's been consistent (after data normalization) regardless of the tuning values including Tb159 mean dual as long as the tuning pass.
Y89: 650-700
Ln115: 1200
Ce140: 2400
Tb159: 1100
Lu175: 1100
Bi209: 500-550
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Jorind

Participant

Posts: 1

Joined: Fri Mar 03, 2023 12:22 pm

Post Tue Jan 16, 2024 9:43 am

Re: CyTOF XT

Hi Yang,

can you tell me what precautions you need to take to get the >80% recovery on the Helios?
What are the most important things to consider when it comes to acquisition?
I will be running a big experiment in February, so I would be happy about tips to maximize cell recovery.

Thank you! :)
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ycheng

Participant

Posts: 4

Joined: Thu Jul 13, 2023 1:35 pm

Post Fri Jan 19, 2024 1:24 am

Re: CyTOF XT

Hi,

The sample introduction system of Helios is quite simple and straightforward, so it's easy to handle when there is a clot. I just carefully and thoroughly backflash (with MilliQ or wash buffer) the loop connected with nebulizer capillary loop and autosampler before and after every run. I also tested out different nebulizer and capillary loop (they are different between different batches), and use the best ones for important experiment. When I see the event rate drop sharply, I stop the run and clean all the loop and resume the acquisition. Yes...I usually staring at the monitor (instead of walking away or using teamviewer) to make sure the acquisition rate is ok, at least for important experiment.
I acquire blood samples at 500K-650K cell/ml, and lower rate for tissue samples, around 350K-500K cell/ml.
Hope this helps.

Yang

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