anyone separated lymphocytes from blood? thought i'd done everything properly then came to defrost them from nitrogen... and gone!!! any ideas?

should beable to do it using a density gradient. have a look at something like Optiprep made by Sigma i think. Can double check tomorrow if that sounds useful.

i used Ficoll-paque, i saw a pellet after i'd collected white layer, but soemthing went wrong after that i think, froze them slowly in FCS with 20%DMSO, more advice would be greatly appreciated

When you say gone, do you mean there are no cells at all (clear medium) or do you mean there is a lot of cell debris? You should be able to check before you freeze down if you have any cells present and their viability. Usually if cells were present when you froze and were killed by the freezing process you would expect to see some cell debris floating aound (I do at any rate, but these are not lymphocytes).

I havent frozen down lympohcytes from blood (I usually use them all). The cells I do freeze down I usually put in their culture medium with 10% FBS and 10% DMSO (ie just add 10% DMSO), but these are cancer cell lines. I usually get pretty good survival that way. How do you freeze them down? I put cells on ice, then into the -80 and then into liquid nitrogen.

hi hun, a person you may want to email is rachel airley at liverpool john moores. she works with alot of lymphocytes/lymphoblasts etc and may even offer some good suggestiuons

How did you defrost them? If you do it too slowly the DMSO can kill the cells apparantly.

HiJen,i defrosted them quickly in water bath them pit straight in fresh medium,
i looked at lymphocytes i'd frozen a few months ago and i could see cells but only a few,i think its going to be better to use them as soon as isolated instead of freezing them, oh and i was freezing themin 20% DMSO? could that be the problem?

To much DMSO can. I remember when I first started and misheard the postdoc. I put all my cells into 100% DMSO. Seemed strange at the time, but I had only just started out so felt I should just go with it. Oddly enough all the cells died. Though 20% should be ok.
You could try 10%, but I would be surprised if that did anything.

Maybe you just dont have many cells/vial you are freezing. Do you count and put a set number in? Maybe its too low (esp if viability is very low as well).

I have to say though, if you are not seeing much in the way of cell debris it seems likely that you are somehow not putting any cells in in the first place. Cells die, but they usually take a while to disappear, and the cell debris is likely to be maintained by the freezing, so you should see lots of bits of cells (though i suppose this depends on how they are being killed). What steps are you doing between the pellet and the freezing?

Hi Richmond,
After i've pelleted the cells i resuspend in FCS alone, then FCS + 10% DMSO. then i wrap in cotton wool in polystyrene box and freeze in minus 80. i move the vials to liquid nitrogen 3 days later. i'm isolating neutrophils from only 1 ml of mouse blood so hard to get good separation of lymphcytes with Ficoll-Paque. thanks everyone for the advice, looking forward to 'starting all over again Monday'.....

oh and i didnt count them before freezing, i assumed there wouldnt be many anyhow

I freeze my cells in 8% DMSO 92% FCS. I have had the same problem, I got them out of the liquid nitrogen and only have a small number of cells surviving.

We usually freeze them gradually in a 'Mr Frosty' (from Nalgene i think) - a controlled cooling container you fill with isopropanol that when put into -80 freezer reduces the temperature at a rate of 1 degree per min before transferring them into liquid nitrogen.

thanks Lis, will ask around department to see if we have this equipment