Hello dear colleagues. Im trying to completely eliminate platelets from blood samples and found a method with using serum tubes for blood collection followed by ficoll centrifugation.
I tried it in this way:
1) collected fresh blood in serum tube
2) centrifuge it shortly after at RT for 5 min at 1500g
3) discarded the serum/plasma and collected blood under the gel (whole volume of blood is uncoagulated)
4) layed it on ficoll 1,077 (blood immediately moves to the bottom of the tube, and it seems to be a problem) and centrifugated at RT for 40 min at 400g
No buffy coat is formed after centrifugation, and no leukocytes are vissible in ficoll layer in microscope.
Did anyone tried smth similar or have some ideas? Thank you!
I have never isolated PBMCs without having the addition of an anticoagulant.
If the cells layered onto 1077 (which should be Histopaque, not Ficoll, different product) is sinking, either you are layering incorrectly, or your cells are clumping due to the fact that there are no anticoagulant in the serum tube. (I have had a resident "layer" incorrectly before, because he didn't understand how to do it correctly. So... assuming that you know the proper technique, I would assume that the cells are sinking because they are clumping togehter.)
My protocols have always collected blood in some sort of anticoagulant, ETDA was the most common, but I've also done citrate. You can try to spin at a lower g (StemCell suggest 150g(?) for 10 minutes without break) during the wash steps. And you will need to make sure that you wash in the 50mL tubes, because you need a LOT of wash buffer volume.
Hope this helps.
Hi, I think you lose WBC in the clot. In my opinion it useless to centrifuge, discard the serum and then use the blood under the gel.
You have to collect the blood in the EDTA tubes as suggested above. If, for any reason you use the serum (wrong) tube, you can immediately aspire alla the blood with a pipette, layer it (dilute or not in PBS, depend on your purpose) on Histopaque and follow the datasheet. If you can avoid the canonical 30 min to clot the blood to obtain serum, maybe you can have a possibility to recover that patient sample.
After you obtain the buffy coat, as suggested in the previous comment, you have to add more wash step, at low speed, to obtained WBC cells without platelets
Yes, you can do it. But you will have a low quantity of the WBC when you are compared to wait for the buffy coat formation.
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