Dear all,
I am planning to do a immune response study in pigs and I have a doubt that I hope someone can clarify me:
I need to get blood from the pigs and then extract serum and PBMCs (Peripheral Blood Monocytes cells) from the blood. Does anyone know whether I can freeze the blood and make up the serum and extract the PBMCs after freezing the blood?
If someone can clarify my doubt I'd be really grateful.
Thanks in advance.
Iker
You need to isolate the pbmcs from fresh blood. Within 4-8 hours of drawing is best to maintain viability. Also, for serum you will need a separate aliquot of blood to allow for clotting. If what you need is plasma, not serum, you can derive both from the same sample.
There are two way for your problems. First - you collect the blood samples and make two aliquot, one in heparin vial and other one in plane vial. Heparinsied blood can be used for PBMC isolation and other one for serum. You allow the plane vial blood for clotting by keeping room temperature for 1-2 hrs then transfer to 4 degree centigrade for 3-4 hrs then spin and collect serum. Second one is collect blood in heparin and keep it 3-4 hrs at 4 degree centigrade. Collect the upper part of the blood i,e plasma in a separate vile with measuring and add same quantity of medium to the blood of adjustment of quantity where you can purify PBMC. To the collected plasma you add 10% of 10% calcium chloride for allowing it to clot. After clotting you can collect serum. I recommend the first procedure. Second procedure is required where sample is very less and precious.
You must isolate the cells first and freeze them in freezing media (that contains a cryopreservant, generally DMSO). Freezing media works by partially dehydrating cells and secondly by minimizing the size of ice crystals that form. If you don't use it, the ice crystals essentially stab the cells to death from within. Also, proteins precipitate during freezing and altogether this would lead to a goopy mass of lysed RBC, precipitated proteins, and dead cell debris. Even if a few live cells remained, they would no longer be able to be separated on a density gradient. Even storing blood overnight at 4oC greatly diminishes the yield of viable cells.
Dear all,
Thank very much to all of you for your answers, they are absolutely helpful! I am really grateful.
Thank you very much!
Kind regards,
Iker
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