Problem about T Cell Purification

Guy Hermans ghermans at luc.ac.be
Tue Jun 9 02:34:54 EST 1998


In article <6l82lh$kca at ustsu10.ust.hk>, boella at uxmail.ust.hk (CHAN Ella
Wai Ching) wrote:

>  Dear all,
> 
>  I would like to know how to use percoll for T cell purification from 
>  blood !!! 
>  
>  I have tried to set the percoll gradients using 3ml 1.06g/ml, 4ml 1.07g/ml
>  then follwed with 1.08g/ml. I loaded 4ml of heparinized blood over the
>  gradient and centrifuged for 25 minutes at 1200rpm (300 x g), room temp, 
>  no break. However, it was found that the separation wasnt clear and it was
>  quite hard for me to distingulish each cell bands.
> 
>  What kind of gradient should I setup for such kind of purification??  

Instead of using a gradient, I would suggest using paramagnetic beads,
coated with either the anitbody towards the cell population you'd like to
sort out (positive selection), or a pool of antibodies towards all other
cells (negative selection) in a Ficoll-purified PBMC prep.

We isolated mRNA or DNA from positively selected cells - the Dynabeads we
use pose no problem with lysis buffers or anything. Some cells can be
separated from the attached beads after selection as well. Just check
Dynabeads: they're cheaper than  Miltenyi! (No commercial interest at all
- just low on budget)

Hope this helps,

Guy

--------------------------------------------
Guy Hermans, PhD student
MS research Unit             Tel 0032 (0) 11/26.92.07
Dr. L. Willems-Institute     Fax 0032 (0) 11/26.92.09
University Campus            E-mail ghermans at luc.ac.be
B-3590 Diepenbeek
Belgium
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