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Unusual Buffy Coat - (I Think)


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#1 djvan

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Posted 27 August 2014 - 05:36 AM

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. 

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#2 bob1

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Posted 27 August 2014 - 12:49 PM

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.



#3 djvan

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Posted 27 August 2014 - 01:16 PM

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?






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