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

From: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 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@UTSouthwestern.edu

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