Hello all. I am trying to isolate buffy coat from whole blood (sodium citrate), and planning on cyropreserving it for later analysis (~3-5 years). As to what analysis would include, we are leaving it as open ended as we can, but mostly am thinking of extracting DNA (Qiagen or home extraction) and looking at epigenetics.
What freezing media is typically used for this type of storage? Would a standard 90% PBS and 10% DMSO work, and what are the drawbacks.
Additionally, I read about the potentiality of platelets clumping and potentially interfering with the sample. Does anyone have any advice on purifying platelets from the buffy coat on that end? Thank you all very much.
Hello, Jamie Chen
A freezing medium of RPMI with 20% FBS and 1% antimycotic/antibiotic and 10% DMSO is good for freezing PBMCs. We do this in our lab and although we haven't stored PBMC for 3-5 years yet, i think it doesn't really matter. Check this article's Material and Methods section. They use the same recipe, but with 7.5% DMSO.
Article The Effects of Storage Temperature on PBMC Gene Expression
About platelet removal, if you really insist on it, then washing your PBMC several times with PBS and centrifuging for longer times at low RCF should remove most of the platelets. If it helps, we have compared several parameters between 2 PBMC isolating methods (with 1 wash step vs 3 wash steps), among which was Platelet removal too. You can check it out here:
Article Optimization of a Density Gradient Centrifugation Protocol f...
Hope this helps and good luck with your experiments!
Hi Jamie
If your concern is only using DNA from the cells, Why don't you isolate DNA and store them at -20 or -80! It will be stable for years.
This will solve your problem of storing the cells as such.
If you still want to store them the best is 80% culture media +20% FBS and 10% DMSO.
firstly, to minimize the platelet contamination from PBMCsw, best way is multiple washing with PBS at low speed for longer time.
And to cryo-preserve buffy coat, you should use 90%FBS+10% DMSO, but if you only going to extract nucleic acid from cells, Using 90% Complete medium (90% RPMI + 10% FBS) with 10% DMSO may also work.
Few people use lower concentration of DMSO as, DMSO hinders in the downstreaming process (DMSO is know inhibitor of PCR). SO, you may use as low as 7% DMSO.
All the best.
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