Hello! I have done PBMC purification from buffi coat several times and have never had any problems, but now two times in a row I cannot get any layers or there are only two: plasma and all the rest. At first I thought maybe the problem is the sample, but now with different sample I still cannot separate the layers. I'll also add a picture. The protocol is as follows:
1. dilute 15 ml buffi coat in 30ml of warm PBS
2. 10 ml of warmed Ficoll into another tube
3. layer diluted buffi coat over ficoll slowly not to mix them
4. centrifuge at 400g for 35 min without break, acceleration also 0, 20C
So far after that I have always had nice layers so I can separate PBMC, but now I don't have any and we cannot figure out why. We are using swinging bucket rotor and 50 ml conical tubes.
We have tried also two different centrifuges, but still no hope.
Thanks for any advice!
Anni
Hi Anni,
I isolate PBMCs from whole blood all the time.
I strongly recommend using SepMate Tubes, Stem Cell Technologies.
https://www.stemcell.com/products/brands/sepmate-pbmc-isolation.html
it makes PBMC isolation super easy.
if you have 30 ml blood; use two 50ml sepmate tubes.
into the tube add 15ml Ficoll (without creating bubbles).
gently layer the blood (15ml) onto the ficoll containing tubes and spin down for 10 minutes at room temperature at 1200g with no brake (you don't want abrupt deceleration).
then decant the top layer into a separate tube and add wash buffer (medium). Spin down at 400g for 10 minutes and you have your PBMC pellet.
Good luck!
Oz
Hello! Thank you for your answers. We found out that the problem was the Ficoll. The problem was how we stored and warmed the Ficoll. Now the problem is solved.
Anni Lepland What exactly was the problem with the Ficoll? We also currently have issues with our PBMC prep (low yield from whole blood) and one possible reason could be that our Ficoll is expired and probably not stored properly (not dark enough). Thank you!
Hello Katharina! Our unopened Ficoll was stored at room temperature for a year or so and when we opened the bottle, then stored at +4C. But best is to store from the get-go at +4C covered with foil. The fact that it is expired shouldn't affect anything. The lab that lent me their one expired over 3 years ago and still works perfectly.
So keep the Ficoll in the fridge covered with foil and hopefully you will get better results! Also, warm only the amount that you need for your experiment, not the whole bottle.
All the best!
Hi Anni,
Never had an issue before with ficoll. I also had experienced in using an expired ficoll and they worked just fine. I kept unopened bottles in room temperature. Once opened, they will be stored in 4 Celcius. Could you elaborate more on the pre-warming the ficoll? What I have been practising is to let them be in room temperature for a while prior to use. Is it similar to yours? I am interested to know.
Hello Wan! I will take out the amount of cold Ficoll from the fridge that I need and then put it to the +37C water bath and leave it there for 2h or if I'm in a rush for 1h so that I'm certain that is has reached +37C. That's the way I have been trained to do it. If the Ficoll is cold/at room temperature, then it is not able to form proper layers. I hope that helped.
The same happened to me. And I found out the centrifuge was not at g (rcf).
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