[Histonet] Re: Histonet Digest, Vol 47, Issue 17

[Eric Hoy]

Anna-Maria asked:

>   I would like some help concerning the collection/separation of Peripheral
> Mononuclear Cells (PBMCs) from whole blood.
>   If anyone has done it with HIstopaque-1077 of Sigma-Aldrich, I would like to
> know which is the appropriate ratio of Histopaque and whole blood, at what
> temperature is the centrifugation done, how many rpm are used and for how
> long. The procedure mentions to use 15ml centrifuge tubes. What will happen if
> I'll use tubes of 5 or 10 ml?
>   If there is anything else making the separation better I would appreciate
> any comment.
>    
>   Thank you in advance,
>    
>   Have a nice week full of joy in work,
>   Anna-Maria

The method I have used for flow cytometry is as follows:

1)      Pipet 3 ml of anticoagulated blood into a 13 x 100 tube.
 
2)      Dilute blood 1:2 with 3 ml of PBS.  Invert gently to mix.
 
3)      Pipet 5 ml of Histopaque-1077 into a 15 ml conical centrifuge tube.
 
4)      Carefully overlay the entire diluted blood sample onto the
Histopaque. Do not allow the blood and Histopaque to mix.

(One tech with whom I worked would put the diluted blood in the centrifuge
tube first, then use a long-tipped Pasteur pipet to introduce the Histopaque
under the blood sample.  I found that technique more difficult, and could
never demonstrate a difference.)
 
4)      Cap the tube and centrifuge 25 minutes at 1200 rpm.

(This is in a Silencer 2110 centrifuge with a swinging-bucket rotor.  The
RCF is about 200-220 G.  Slow and gentle.)

The centrifugation is at room temperature.  After spinning you will have
four layers: plasma on top, PBMC, histopaque, and RBC/granulocytes.
We remove the plasma with a pipet, and then harvest the PBMC.

Good luck with your cell separations!

Eric

===================================================
Eric S. Hoy, Ph.D., SI(ASCP)
Clinical Associate Professor
Department of Medical Laboratory Sciences
The University of Texas Southwestern Medical Center
Dallas, Texas
Email: Eric.Hoy <@t> UTSouthwestern.edu
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