Hi Sophia,
In our research facility, we almost always isolate plasma from whole blood prior to PBMC isolation, since we often use the plasma for other tests. Normally, after removing the isolated plasma, we will replace the volume back to the original volume using buffer (PBS). The point of this step is to simulate the original volume of whole blood prior to your dilution. Then, you may proceed with your PBMC isolation by making the appropriate dilution with PBS.
Hi,
I just encounter the same question and was wondering if the solution proposed here was working as expected?
If I understand correctly, once you took the plasma, you:
1) replace the volume of plasma with PBS to reach the initial blood volume.
2) dilute this volume again 1:2 as usual in a Ficoll centrifugation protocol.
would this procedure be right?
We are wondering if the volume of PBS would'nt disturb the grandient centrifugation by altering the density of the blood.
Hi Quentin,
Dorothee Bienzle at the University of Guelph gave me this advice:
Your hares will have about 40% of the blood volume taken up by cells, and about 60% by plasma. So, 3 ml of blood (which is quite a bit) will yield about 2 ml of plasma, if you spin the sample hard. If you take out all the plasma, and replace it with phosphate buffered saline, you change the buoyancy properties of remaining cells on Ficoll quite a bit, and I would not count on this still working. If you are only taking 0.5 to 1 ml of plasma, and then reconstitute the sample back to about 3 ml with saline, the Ficoll gradient should still work. In general replacing about 50% of plasma with saline preserves the buoyancy of PBMC's.
So I switched to taking a smaller volume of plasma followed by the ~1:2 PBS dilution and all was well!
Dear Sophia,
Thank you very much ! I also noticed a difference when taking too much plasma.
I will try as you proposed, thanks for the advice!
Regards
Hi I am very interested in this question. We would like to collect both plasma and PBMC from the same sample. I would like to know more detail about the protocol
1. if you want to collect both of them, do you first collect blood sample in an EDTA tube or heparin tube?
2. Do you centrifuge your blood sample at 2000g ? will this effect the survival of your PBMC or cause any coagulation? or should I use less g to centrifuge blood?
Thank you very much
Regards
Hi,
for the tube I'll let people who may have tried both answer, but any of these 2 should not bring any difference.
for the centrifugation, i first centrifuged my 10ml tube of blood at 800g for 10 min; i was usually able to collect more than 1ml of serum. However, you will notice a high variation depending on the sex of the donnor, and on their level of hydratation. if you want plenty of serum, I advice you to recommand your male subjects to come well hydrated to give blood.
then just replace the volume of serum with PBS to go further with the experiment; however, i only took 1ml of serum in my experiments; I don't know if more serum taken away and replaced by PBS might influence the quality of the Ficoll step.
good luck!
Quentin
Hello Quentin
Thanks for your suggestion.
So 800 x g is good enough for you to collect plasma without destroy cells? because a standard protocol for plasma collection requires centrifuging the whole blood at 1000 - 2000 x g for 15 min. I am always wondering can I still collect PBMC after this step.
Regards
yes I was concerned about a too hard centrifugation as you are; that is why 800g appears as a pretty nice compromise. Your blood cells will not be highly compacted, therefore you'll loose a bit of serum. However, this will make it so that your cells are fine afterward,
If you really are concerned about your cells, you could also remplace the serum with PBS supplemented with FCS, to reduce the stress.
Finally the fact to not be able to take all the serum away might be a good thing, since too much PBS dilution might disturb the Ficoll efficiency.
good luck,
Quentin
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