In our lab, we isolate PBMC from undiluted whole blood using Histopaque. This works perfectly when layering 3mL undiluted whole blood over 3 mL Histopaque. The problem is when working with larger volumes of blood the process of layering 10-15 (15mL) tubes at a time becomes very time-consuming. Does anybody use Histopaque and undiluted whole blood in 50 mL tubes? What volumes of undiluted whole blood and Histopaque do you find gives good clean PBMC separation?
Thank you,
Thomas Byrne
Hi, I use Limpholyte instead of Histopaque but if yours is 1,077g/ml I think is the same. I had more experience with diluted blood but I try undiluted for RNA extraction from PBMC.
9ml of limpholyte and 9ml of blood in 50ml tube, ratio 1:1. Honestly I always prefer 15ml tubes, the ring is better, but if you have a lot of patients the number of tubes increased a lot.
I used an Eppendorf centrifuge 5810R with normal acceleration or 4 and brake 5 or 0. I don't know what is your centrifuge so I recommend to use your own blood to make some test.
From my experience with diluted blood in 50ml I tried this:
10ml limpholyte and 10ml of diluted blood with PBS (5ml + 5ml PBS, ratio 1:1)
15ml limpholyte and 16ml of diluted blood with PBS (8ml + 8ml PBS, ratio 1:1)
You have to improve the PBMC ring according with your centrifuge, with undiluted blood can be a little tricky
Valeria
Hi Thomas:
I tried both 15ml and 50ml tube for PBMC purification from diluted and undiluted whole blood, all worked well. There are a few tips which i think they matter:
1) If the Ficoll or other buffer stored in fridge, restablish their temperature to RT;
2) Centrifuge speed of 270 g around, acceleration and brake to 0;
3) PBS without Ca Mg wash, centifuge speed and time are important for cell yield, purity and aggregate, need to calbrate on individual machine.
4) After decant wash buffer, finger flip the tube to resuspend cells can avoid aggregates.
Best regards
Shiqiu
I have worked with both diluted and undiluted blood. For undiluted blood I kept histopaque:Blood ratio 1:2, while on diluted blood ratio was kept 1:3 for good and pure PBMCs in 15 ml tubes. I centrifuge the tubes with no break.
I think in 50ml tubes with undiluted blood you may use 1:2 ratio of histopaque to sample.
If you don't want to dilute the sample there is a suggestion. You may remove the plasma and add equal volume of PBS. This will be helpful in reducing platelet contamination.