Unusual Buffy Coat - (I Think) - (Aug/27/2014 )

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?

Thanks!

P.S. I apologize for the quality of the video - I was try to hold my phone and pipette at the same time. 

Looks like you have lysed the white blood cells and caused the DNA to aggregate. Though this would usually present as a more of a sticky goo than the disk like object you seemed to pull up.

bob1 on Wed Aug 27 20:49:39 2014 said:

Looks like you have lysed the white blood cells and caused the DNA to aggregate. Though this would usually present as a more of a sticky goo than the disk like object you seemed to pull up.

Spinning at 2000xg would be the cause of the lysis?