Summary of replies to : How do you freeze cells for storage prior to staining? To all those who also wanted to know the answer to this question, these are the replies I recieved, and to those who sent information, thankyou very much. Sorry if I missed someone out, not intentional ........................................................................................................................................................... As far as I know there are no cons. We have done this for intracellular staining for 6-7 years on a bizillion samples without a problem. There does not appear to be any added noise due to the freeze-thaw. After fixation the cells are diluted in a 4 fold excess of PBS with 0.1% BSA and then centrifuged at 2600 rpm (approx. 750 x g?) on a table top Sorvall cell culture centrifuge. The pros are that you can freeze down replicates and : 1. be able to disassociate the stimulation from the staining experiment; 2. can batch samples stimulated on different days; 3. have room for error; 4. be able to look at additional cytokines and markers after your first experiment or even years later. Additionally, we perform all of our single color controls using unstimulated fixed PBMC. We fix a billion or so PBMC and then aliquot 10 million to a tube. Comes in very handy for compensation as well as for titration experiments for cell surface markers. We use PBS with 10% DMSO. Don't need to use fancy cell culture grade DMSO, as the cells are dead! ............................................................................................................................................... Dear Heather, I successfully used the protocol reported in Elson et al. Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2, and T helper 0 cells within the human CD4+CD27- lymphocyte subpopulation. J Immunol. 1995 May 1;154(9):4294-301. ................................................................................................................................................. Dear Heather, we routinely stimulate whole blood with PMA/Ionomycin for 4.5hr then add 2ml of BD FACS lysis solution. The diluted blood is incubated at room temperature for 10min then transferred directly to -80oC. When we are ready to process for intracellular cytokine staining we return them to room temperature until fully thawed and then continue with the BD protocol. We have compared staining freshly stimulated to freeze/thawed cells and there is no significant difference. what we do is... 1> Stimulate the whole blood for 4.5hr 37oC 2> Add 100ul of stimulated blood (no need to wash as the lysis buffer, permeabilization buffer and washes in between are enough to remove the majority of RBC by the end of the preparation) to a FACS tube 3> Directly add 2ml of x1 lysis buffer (x10 stock in deionised water, Becton Dickinson)and leave at room temperature for 10min. 4> The sample can then be transferred directly into the -70oC for storage. In answer to your other questions , I would not add lysis buffer to 1ml of blood only because the time it would take to defrost the sample. I have tired something similar and the cells seemed OK but I have not tested this extensively. I would adjust the concentration of the lysis step if you intend to do this only because the buffer also contains a fixative required for satisfactory permeabilization. We used to surface stain at room temperature for 10min prior to adding the but on testing staining before and after there was little difference so for ease of handling and storage we routinely surface stain and intracellularly stain at the same time. ........................................................................................................................................... BD themselves also replied, thankyou, their procedure is as outlined above in one of the replies I recieved. ............................................................................................................................................................. It was also suggested that I search the group's archive . . . < http://www.cyto.purdue.edu/hmarchiv/index.htm > As the question of analyzing frozen cells has apparently been discussed several times - - ..................................................................................................................................................................... Hope this is helpful to you Heather
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