tissue storage

[fery]

Dear Friends

I would like to know what is the simplest way for storage tissue samples(polyps and biopsies)   for a period ot time ( up to 3 months)  for  flow cytometry analysis. I know that the best way is using cryomedia (RPMI + DMSO ) and freezing overnight in -70 and then transfer to liquies nitrogen but what happen if I put my samples immediately in liquid nitrogen or store them for long time in -20 freezer.
any comment or suggestion would be very appreciated.

Thanks in advance

Yours

Fery

[Pavel]

If you put them directly into liquid Nitrogen the cells will most likely lyze (burst). Ideally, gradual freezing is best because it helps cells to freeze slowly not damaging the membranes too much. They even have automated machines for doing that when it comes to saving blood cells for a later infusion.
In my experience though, any type of freezing cells ends up in losing a bunch of them. So it is worth doing it as carefully as possible: first on ice, then -20 for a couple of hours, then -80 overnight, then liquid N2.

When stored at -20 degrees, some biomolecules will still be degrading.

I would suggest fixing them instead, for example, in formalin.  
 

[fery]

Dear Pavel

Thank you very much for your very valuable comment ,  but as I mentioned I would like to do flow cytometry analysis so I can't fix my samples. I appreciate you very much if you please let me know about the methods that I can use instead of -70 freezer, I have heard that if I put  cryovials in isopropanol and transfer it to liquid nitrogen , it will protect samples from sudden freezing but I am not sure about it.

Thank a lot

Yours

Fery

[Pavel]

What kind of flow analysis?

For example, we do flow for DNA content a lot, and for that we fix cells in 70% ethanol and store them up to a few weeks in -20 freezer.

Many proteins should also be ok in formalin or paraformaldehyde. It depends on your antigen and antibody though, a lot. But still sometimes you can just store them in formalin without much increase in background. I also heard recommendation of fixing them quickly and then storin in a mild sodium azide solution in PBS.