Replacement of "old" 3H-thymidine assay.

Hua-Chen Chang hua-chen.chang.1 at purdue.edu
Mon Mar 29 12:21:49 EST 1999


I like to get some advise from all the experts on immunology.

I am trying to evaluate lymphocyte proliferation with speficic antigen
stimulation.  The antigen is a virus.

I noticed that most of the articles use inactivated virus for this assay.
However, I have heard that live virus can also do the work.  The reason for
inactivated virus for stimulation in lymphocyte proliferation is to provide a MHC
class II processing pathway or avoid the cell killing by the virus, or any
particular reason?

Does MHC class II processing pathway is still required for in vitro lymphocyte
proliferation assay?  CTL assay by standard Cr-51 release assayt does not need a
live virus for stimulation.

Is APC required for nonspecific stimulation with ConA in this assay?

It only takes two days for ConA stimulation.  How many days does this assay need
for antigen specific stimulation?

Thank you all.
Hua-Chen Chang

Schrum wrote:

> If you have access to a flow cytometer, you can use CFSE (Molecular
> Probes).  It is a green dye that can uniformly label lymphocyte plasma
> membranes, and it dilutes in half each time a cell divides.  Nondividing
> cells remain labeled virtually indefinitely in cell culture, and CFSE
> labeling has also been used to track cells and examine their
> proliferative history in vivo.  Plus, it's a cinch to use.  See Wells,
> et al., Journal of Clinical Investigation 100: 3173-3183 (Dec 1999) for
> description of labeling procedure.
>
> good luck,
>
> --Adam Schrum
> laboratory of L.A. Turka
> Univesity of Pennsylvania
>
> Jens Rainer Hansen wrote:
> >
> > I want to replace my 3H-thymidine assay on human lymphocytes, since it is
> > to hard to perform when having a lot of samples.
> >
> > I have tryed alamarblue but it doesnt work with lymphocytes.
> > The MTT assay should also be insensitive with these cells.
> >
> > I have also tryed Brdu incorporation it seems ok, some times, but since the
> > lymphocytes are non adherent it is nessesary to dry the cells on the
> > microtiterplates with an hairdryer, and this procedure was not stable for
> > me.
> >
> > Do you have experience with an easy assay to meassure DNA synthesis/cell
> > proliferation on human lymphocytes (non adherent) using other methods than
> > 3H-thymidine, or methods where it is easy to handle the samples using
> > 3H-thyminine, that means not punching filters into vials etc.
> >
> > Yours Jens R. hansen




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