We would be collecting blood samples from some extreme climatic conditions and would require atleast 6 months before I can bring it to the lab. Please can your kind selves suggest the best method to collect, process and store the PBMCs provided we will have only -20C.
As a routine in our lab we collect blood in EDTA / sodium citrate tubes. With ACK lysis solution lyse the RBCs, wash the leucocytes with PBS twice and preserve it with 10% DMSO and RPMI. Please suggest to improvise this.
Thanks in advance
Dear Niklas, thanks for the answer. i will using the pbmc for telomere length and telomerase estimation. and we
Hi Ragavendrasamy,
I've good experience by using both Heparin and EDTA for PBMCs.
Use Ficoll density gradient medium for layering the sample and centrifuge it @ 400 xg for 30 mins and collect the buffy layer and proceed the PBS/HSBS/plain RPMI wash for 2 time.
And its not necessary to use ACK lysin, since we found poor viability while using it.
For storage, better prepare the CPS with 80% of FCS + 20% DMSO. Suspend 1 million cells/ 100uL of CPS. And -20°C is not suggestible for PBMC long time storage. You must transfer to -80°C for 6 months and liquid nitrogen (-196°C) for more than 6 months.
- Kadar Moideen Abbas
Thanks Dr Moideen, Thanks for sharing your valuable inputs. However, the limitation here is that we will not be having access to -800 C freezer. Is it ok to store it at -20 for atleast 3-4 months period?
And, what shall be the maximum time that i can store the whole blood before i can use it for separating the PBMCs? I am asking this to ensure to have myself prepared.
Thanks again,
Actually, I'm afraid to suggest you go with -20oC. But, if it is your situation,
1. You must be aware about power fluctuation for the freezer, so that the temperature can be maintained at -20oC, otherwise it will lead you to the loss of samples.
2. Transfer the PBMC samples to Dry Ice from the freezer as soon as possible during the transportation. Delaying will surely affect the viability of your samples.
And, about 6 hrs you can have the whole blood at room temperature. If you take more than 6 hrs, better you keep it at 4oC.
Thank you.
Thanks very much Dr Moideen.
I was told that there are no power fluctuations as such. Hoping for the best. I will follow your advise
Thanks much
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