Hello Ms. Debbie O'Reilly,

Blood samples were preserved with CPDA or anything else? 

Regards, 

Babu

Hi Debbie, I have not processed frozen blood samples before, but I'm thinking you can thaw the blood, and collect PBMCs using Ficoll gradient separation methods, which can also get rid of the dead cells in the meanwhile. Then you can sort NK cells by flow cytometry with CD56+ markers.

Hope it would be helpful for you.

Jennifer

Thank you,

To answer you Baba, there was EDTA added to the samples and since they are not coagulated I assume some form of anticoagulant.

Han, thank you, I had attempted separation using lymphoprep, and I have CD56 microbeads ready to go, but separation did not occur. 

Any ideas?

Of course, the cells (total PBMCs isolated) should be resuspended in freezing RPMI medium (50% FCS and 5% Me2SO) to 10-25 x10 millions/ml final concentration at room temperature (cryobials) and finally cryopreserved at -80˚C as routine support.

Respect of the specific cells isolation from PBMC, you can check by immuno sorting, when is more adecuate and practical, before or after of cryopreservation.

Regards

Alfredo

Hi Debbie, the NK cells within PBMCs should be around 15%, not too much. How many volumes of blood you performed? If not many, sorting might be much better and easier to obtain pure NK cells than using microbeads.  

Thank you all, advice much appreciated. 

Yes Han the volumes of blood are small, so I will consider sorting. 

Hi Debbie,

I am having similar situation. My samples were frozen without DMSO ( and probably with EDTA). Just wondering how do you thaw and isolate PBMCs?

thanks

Hi! Was it possible to obtain live PBMC from the frozen samples?