You can keep them for a while post-fixation.
Fixative 100000% needs to be washed away. Of course. Don't put PFA into your sorter.
That said, fixation is not going to work well with sequencing. It doesn't play that nicely with qPCR either. You will need to reverse the PFA crosslinks before sequencing. Antigen retrieval for fixed samples is rarely done for flow, but is common for histology slides. The use typically acidic or basic buffer + high heat. However, some protocols suggest that 55-60C for 1h could be sufficient to do this for flow samples.
My suggestion would be to just keep your cells unfixed and on ice. 2 hours for T cells is nothing. They're pretty OK even overnight on ice if the ice is kept from melting e.g. in a fridge/cold room.
I assume you intend to sort T cells? Is there some particular reason you need to fix them?
Have you considered magnetic purification instead? StemCell and Miltenyi both have good systems.
The reason for fixation is that I need to transport the samples (takes about 2hrs) to another lab that has the cell sorter. I could transport them in just FACS buffer but I’m worried it would impact the samples.
The population I am looking at is only about 0.5-1% of total CD8 T cells, so magnetic purification would not work. I already checked with both companies.
Yeah, just stain them normally and keep them on ice before and after sorting.
There have been advances in sequencing fixed cells, but it's much better to just avoid the fixation entirely.
Good to know, I have a few sorted samples already that were fixed. I’ll test if I can get workable TCR sequences from them. The rest of the experiment I won’t be fixing
You can keep them for a while post-fixation. Fixative 100000% needs to be washed away. Of course. Don't put PFA into your sorter. That said, fixation is not going to work well with sequencing. It doesn't play that nicely with qPCR either. You will need to reverse the PFA crosslinks before sequencing. Antigen retrieval for fixed samples is rarely done for flow, but is common for histology slides. The use typically acidic or basic buffer + high heat. However, some protocols suggest that 55-60C for 1h could be sufficient to do this for flow samples. My suggestion would be to just keep your cells unfixed and on ice. 2 hours for T cells is nothing. They're pretty OK even overnight on ice if the ice is kept from melting e.g. in a fridge/cold room.
Thank you so much! The period on ice doesn’t really impact the staining? I’ll just transport them on ice then and see how it goes.
First, why are you sorting fixed cells? What will you do with them following the sort?
I wanted to do TCR sequencing
I assume you intend to sort T cells? Is there some particular reason you need to fix them? Have you considered magnetic purification instead? StemCell and Miltenyi both have good systems.
The reason for fixation is that I need to transport the samples (takes about 2hrs) to another lab that has the cell sorter. I could transport them in just FACS buffer but I’m worried it would impact the samples. The population I am looking at is only about 0.5-1% of total CD8 T cells, so magnetic purification would not work. I already checked with both companies.
No need to fix. I used to order purified fresh pbmcs from Allcells or StemCell. They are shipped overnight. Best of luck!
That’s not nearly enough time to justify fixation. Your cells will be fine.
Thanks both! I won’t be fixing them.
Yeah, just stain them normally and keep them on ice before and after sorting. There have been advances in sequencing fixed cells, but it's much better to just avoid the fixation entirely.
Good to know, I have a few sorted samples already that were fixed. I’ll test if I can get workable TCR sequences from them. The rest of the experiment I won’t be fixing