Dear researchers,
I have isolated PBMCs from whole blood using EDTA tubes with Lymphoprep. I followed the protocol, but I have come across some forums suggesting the dilution of blood with RPMI, HBSS, or PBS. I opted for PBS, but after centrifugation, I obtained a very thin layer of PBMCs. I am uncertain about what went wrong. The protocol indicates a blood-to-Lymphoprep ratio of 1.5:1, but some forums suggest a ratio of 1:1. Which ratio is recommended?
Can you please provide guidance on the choice of diluent (RPMI, HBSS, or PBS) and clarify the optimal ratio for isolating PBMCs?
Thank you for your assistance.
Hello Hind Sebbah,
While isolating PBMCs from whole blood there are some points you should keep in mind.
1. Use diluted blood for PBMC isolation. Diluting blood reduces the degree of RBC aggregation as well as freeing trapped PBMCs. If left undiluted, trapped PBMCs sediment with erythrocytes, reducing the yield.
2. The diluted blood is added to the gradient by gently pipetting onto the density gradient medium with the tube held at an angle. To obtain good separation, it is paramount that clean separation of blood and density gradient solution is maintained prior to centrifugation. Do not introduce any air bubbles when adding the density gradient layer as this will have profound effect on subsequent separation.
3. Centrifugation should be carried out at 18-24°C. Higher temperatures (37°C) enhance RBC aggregation reducing yield while lower temperatures (4°C) inhibit aggregation, decreasing purity.
4. Always use freshly isolated blood samples (< 24 hours after collection).
As far as the diluent is concerned, use PBS + 2% FBS. Dilute the blood in an approximately equal volume of PBS + 2%FBS (mix) in 1:1 ratio. Addition of serum will increase the PBMC yield. Also, for high PBMC yield, it is important to maintain the same volumetric ratio of diluted blood sample to Lymphoprep (1:1).
Best.
Malcolm Nobre I would like to thank you very much for your time for explaining to me how to do. I have just one last question: in which tube do you collect blood? In heparin tubes? EDTA tubes?
Best regards
I agree with Malcolm's suggestions. My only concern is about using EDTA tubes for blood collection. We used powdered heparin (5-1 0 mg/20ml blood) to prevent clotting. Alternatively, heparin-coated tubes
are also recommended.
Hind Sebbah, I agree with Professor. It depends on the downstream application. For instance, Heparin is much better than EDTA for the PBMC preservation for downstream B and T cell analysis. For some applications, EDTA may not be the preferred anticoagulant for PBMC isolation.
You may want to refer to the paper attached below for more information.
https://link.springer.com/article/10.1007/s40139-019-00192-8#:~:text=The%20differences%20were%2C%20however%2C%20significant,PBMC%20isolation%20for%20some%20applications.
Best.
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