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Cell loss during acquisition prep

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Jahangir

Master

Posts: 52

Joined: Sun Oct 29, 2017 6:34 pm

Post Thu May 25, 2023 2:56 am

Re: Cell loss during acquisition prep

Hi Pauline,

I echo Mike's reply too. I don't think it's wise to omit the last CAS+ wash because of the reasons he stated - although it *might* be better for that particular run of yours, it won't be for (1) the instrument, (2) the operators cleaning and maintaining the machine and (3) other users who will use it after you.

But I also empathise with you too as my lab and I sometimes experience heavy cell loss in the final washes CAS+ solution. I'll detail the way I perform the final few steps of the protocol below and hopefully give some useful tips:

1) Cells have been incubating in iridium intercalator overnight (or 1+ hour(s) at RT). I add 2 mL of CSB (total solution volume is 3 ml). Centrifuge at 1000 x g for 4 mins*.
2) Remove supernatent. Add in 2 mL of CSB. Centrifuge at 1000 x g for 4 mins.
3) Remove supernatent. Add in 3 mL of CAS+**. Centrifuge at 1000 x g for 4 mins.
4) Remove supernatent. Add in 1 mL of CAS+, filter using a 40 um PET filter into a new tube***, count and adjust concentration of cells to run at around 750k+ cells/mL****.
5) Add 1:5 EQ beads***** and EDTA to final working concentration of 2 mM******.
6) Run on XT.

* I found that, in my experience of running single cell epithelial cells from colorectal cancer organoids which have been co-cultured with fibroblasts, 800 x g sometimes isn't strong enough to pull down the cells stuck on the (inner part of the) sides of the FACS tube throughout the whole protocol. Hence why I've increased this to 1000 x g, which helps retain more cells. I also wash the sides of the tube when initially adding in fresh CSB for each wash. Saying that, the downside would be that it would also cause more debris to be carried forward. Just to add, the cells I've been using have been fixed using 4% PFA, so I believe they have the integrity/strength to withstand the higher centrifugation speeds.

** I only wash once with CAS+ solution, but instead of washing with 2 mL, I use 3 mL and properly resuspend the cells into it. I find this is a good compromise between retaining cells and preventing cell loss alongside preventing build up of salts from the residual CSB in the tube. My rationale behind this is that tubes which have been coated with a protein buffer (such as CSB, FBS, etc) help cells pellet during wash steps. So the first wash with CAS+ solution will still have *some* residual CSB left in the tube, which will allow cells to pellet. But additionally, the 3 mL of CAS+ should remove the bulk of the residual CSB. I haven't personally noticed any build up of any salts in the nebuliser or cones with the above.

*** My lab and I use this filter instead of the nylon 35 um filter as it's a stronger filter and therefore does not stretch as much when pipetting my large, 'sticky' cells through it. Therefore, the only cells that should get through *should be* around 40 um in size. And once they've been filtered into a new tube, I *never* centrifuge/wash them thereafter, doing that will cause huge cell losses due to the rationale above.

**** Again, in my experience, I don't dilute my sample too much as this is one area where I've seen with my colleagues too, that diluting the sample heavily just before running (to prevent cells clogging the nebuliser or otherwise) causes the event rate to start significantly low which only gets lower as the acquisition proceeds. And therefore instead of the expected 30-50% cell loss that occurs normally during the acquisition, I observe a 70%+ loss, which mostly happens when the sample has been heavily diluted prior to running. So I try to keep it at around 750k cells/mL to 1 million cells/mL.

***** the 1:5 EQ beads is something I've always done, esp with the Helios. The rationale behind this was more EQ beads won't harm your sample, whereas less might cause a few parts of your run to be removed due to insufficient amount of EQ beads in each section of your sample being normalised.

****** 2 mM EDTA is in all of our wash buffers, CSB and CAS+ and in the acquisition too. As this helps our very sticky cells to stay as single cells and prevents them from clumping together. For my lab and I, this is a must.

Hope the above helps!

Best regards,

Jahangir
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PaulineM

Participant

Posts: 14

Joined: Thu Apr 12, 2018 1:05 pm

Post Thu May 25, 2023 6:09 pm

Re: Cell loss during acquisition prep

Thank you very much for your reply :)

If I can bother you with an extra question:
Have you ever attempted to perform a final rinse in water before running samples in CAS+ (for extended acquisition time)?
I wonder whether the water would be diluted enough when resuspended in 3ml CAS+ for the signal quality to be maintained over the course of the acquisition.

Pauline
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PaulineM

Participant

Posts: 14

Joined: Thu Apr 12, 2018 1:05 pm

Post Thu May 25, 2023 6:23 pm

Re: Cell loss during acquisition prep

Sorry Jahangir, I've seen your reply after my reply to Mike.
I thank you so much for all the details that you have shared.

I'll definitly have to try your procedure. I'll let you know about the outcome, especially for the 1000 x g centri, which scares me a little ;)
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PaulineM

Participant

Posts: 14

Joined: Thu Apr 12, 2018 1:05 pm

Post Wed Jun 07, 2023 1:39 pm

Re: Cell loss during acquisition prep

Hello Jahangir,

Just to let you know that after running some tests, I confirm that, as you said, it was the centri after the cell filtration into CAS+ that caused the important cell loss that we experienced.

I thank you for your help
Pauline
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