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
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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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