Actually I have never done such experiment, but I was thinking to do
something like this. CFDA,se staining would be a choice for
proliferation assay, but I know the best results can be obtained it is
to dessect the animal one week after T-cell injection, It is still
visible after 3 weeks, but the non-radioactive thymidine analog
bromodeoxyuridine (BrdU) can be added to the the culture, or inject to
animals.
Actually I am also seeking the advice to how to do exactly. Cheng
On 14 Nov 2002, Stacy Ferguson wrote:
> Date: 14 Nov 2002 12:56:32 -0800
> From: Stacy Ferguson <stacyf at stacyf.net>
> Newsgroups: bionet.immunology
> Subject: High throughput T cell isolation/proliferation?
>> I'll be starting an experiment that requires to me immunize 125 mice
> and do T cell proliferation and cytokine assays on splenic T cells
> from all of them. I can't pool the groups. I've done all of the above
> in the past, but never with so many samples. I expect the nightmare
> in this experiment will be purification of T cells put into the assays. Does
> anyone have suggestions for the most efficient method for isolating
> T cells from 125 populations? I can space them over a three day
> period since I have three main groups (three antigens) but I don't
> want to subdivide those groups because I'm comparing different vaccination
> methods and adjuvants for each of the three. I'm on a deadline schedule
> that won't let me try each antigen in separate, more managable experiments.
>> Also, the company I'm at doesn't yet have a radiation license. I will
> need to use a non-radioactive method for proliferation assays. I'm planning
> to use a formazan based detection method but I'm amenable to changing
> that. I've only used radioactive methods in the past and my impression, at
> least
> based on literature with associated kits, is that contaminating non-T cells can
> add
> a lot of noise to the assays. In my mind, panning might be a good
> high-throughput
> approach to isolate CD4+ cells out but it's not as clean as other methods.
> Should
> I expect this to be a huge issue for me with the formazan-based detection? Also,
> if I use a pan-T isolation method, will CD8 cell contamination be a problem in
> these assays?
>> Thanks in advance,
> Stacy
>>
--
Dr. Cheng Luo
Medicity Research Laboratory, Biocity 4th Floor
Turku University
Tykistökatu 6
FIN-20520 Turku
Finland
Tel. 358 2 3337025 or 7038(Lab)
Fax 358 2 3337000
Email Cheng.Luo at utu.fi
mobile 050 3506158
Tel. 358 19 5244448(home)
http://users.utu.fi/cheluo
