For my project I primarily needed PBMCs which I extracted from whole blood using BD vacationer cell prep tubes. At the time I also saved the plasma fraction. Now I have reason to interrogate the plasma for TGFB1 which requires platelet depleted plasma. The CPT manual suggests that the platelets would have fractionated with the PBMCs, leaving the plasma relatively platelet poor, but obviously this is not the standard way to achieve platelet poor plasma. Does anyone have any ready info on the platelet content of this plasma? I can't assess the frozen archived samples without the platelets degranulating obviously, so I may check using fresh samples, but wondered if there was experience out there before taking any more of my own blood!
Hi Priya, I dont think its a big issue for your purpose. What was the centrifugation speed for PBMC/Plasma isolation ?
You can thaw plasma slowly at RT, this sample may have platelet and other cell derived microparticles, spin some of the plasma at 20,000 g for 10 min (to remove MPs) and compare its TGFb elisa with non-centrifuged sample. If you have access to flow cytometer, you can also check the platelet/microparticle content via FC method.
Best of luck
Hi - thanks for the info. The samples were spun for 20mins at 18rcf. While I'm waiting for data from the manufacturers, I propose to do exactly what you say....
Isolation of mononuclear cells from whole blood is a first step for many in vitro assays. One currently accepted technique for mononuclear cell separation, referred to as the FICOLL™ Hypaque™ method, employs a liquid density gradient medium of FICOLL™ 400 and sodium metrizoate or sodium diatrizoate solution (1,2,3). The procedure uses anticoagulated blood, collected by routine phlebotomy, which is diluted with a buffered solution, and then carefully layered onto the medium. This preparation is then centrifuged to isolate the mononuclear cells above the medium. The cells are harvested by carefully pipetting them from the liquid interface. The BD Vacutainer® CPT™ Cell Preparation Tube with Sodium HeparinN combines a blood collection tube containing a sodium heparin anticoagulant with a FICOLL™ Hypaque™ density fluid and a polyester gel barrier which separates the two liquids. The result is a convenient, single tube system for the collection of whole blood and the separation of mononuclear cells. The BD Vacutainer® CPT™ Cell Preparation Tube with Sodium HeparinN reduces the risk of sample contamination and eliminates the need for additional tubes, pipettes, and reagents. Samples can be transported without removing them from the tube
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