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Re: Methanol fixation of cells

Posted By: ojasvichaudhary, on May 9, 2018 at 8:41 AM

Great job 

Re: Methanol fixation of cells

Posted By: long, on Jun 22, 2018 at 12:36 PM

Please add me: huansci@gmail.com

Re: Methanol fixation of cells

Posted By: mstreube, on Oct 19, 2018 at 7:30 AM

Dear Dr. Chen,

dear 10x Users,

 

We just recently discovered your very interesting and useful paper about the Methanol fixation of hPBMCs which can be used for 10x Chromium Controller.

 

One general questions to you and also to other 10x users and two in terms of cell clumping:

 

1) In this paper PBMCs were stored after fixation at -20°C or -80°C for up to 3 month. Could anyone see  differences between these temperature conditions in terms of gene expression?

 

2) Did anyone observe any clumping PBMCs during the procedure (fixation or rehydration)? In our last experiment we observed cell-cell adhesions after the fixation. Instead of 2:8 PBS/BSA and Methanol ratio (like for Chen et al. 2018) we did 1:9 (like its mentioned in 10x protocol) and we doubled the amount of Methanol, because we wanted to fix 11 Mio total cells. In 10x protocol cells are only resuspended with 1xDPBS (not including 0.04% BSA). Can this lead to cell clumping?

 

3) Do you keep the cells on ice for the whole procedure (beginning of methanol fixation part till end of rehydration? In 10x protocol its mentioned that too long storage on ice for reyhdrated cells can lead to cell aggregation and clumping.

 

I would be very glad for any kind of advice and help, thank you.

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Re: Methanol fixation of cells

Posted By: shauna-10x, on Oct 22, 2018 at 1:54 PM

Hi,

 

This reply is forwarded on behalf of Dr. Chen from the 10x Community email account.

 

Dear Michael:

 

Thanks for your interest in our paper.

 

Here are the answers to your 3 questions:

We did not see differences of RNA quality between these two temperatures. But we did not look at the gene expression of samples stored at -80C.
After fixation, the cells are sticky. BSA is suggested to be added to the resuspension buffer. We keep cell concentration at less than 1 million/ml fixation buffer (PBS-buffered methanol) to avoid clumping.
We resuspend the cells right before loading to the chip. The cells are always kept at 4C.

Hopefully it helps. Let me know if you have any other questions.

 

Best,

Jinguo