Hello. I was interested in isolating PBMCs from a large volume (800mL) of heparinzed canine whole blood. Instead of using an obscene amount of Ficoll, I read online that you could isolate the buffy coat, dilute it 1:1 with PBS to prevent aggregation, and then apply that to the Ficoll layer. However, I had an unusual looking buffy coat - I've attached a video.
Here's what I did:
1) Blood was brought to me post operation - less than 30 minutes ex vivo.
2) I separated the blood into 50mL conicol centrifuge tubes, and spun at 200xg, RT, for 10 minutes - no break. No buffy coat was visible.
3) I spun a second time, this time at 2000xg, RT, 10 minutes - no break. I took the tubes out, and what you see in the video is what came out of the centriuge.
In my previous experience, the buffy coat has been a layer, and I could easily draw it off with a pipette tip. I tried that with this, and it would end up pulling the RBCs right through the "buffy coat". So, I switched to a wider mouthed, standard pipette. However, I spun in a different centrifuge before, and you could only set the break to "slow" - perhaps that was causing the buffy coat to separate more, and form a layer, as compared to what we see here?
So, can anyone confirm that this is the buffy coat, or tell me what it is instead?
P.S. I apologize for the quality of the video - I was try to hold my phone and pipette at the same time.