Where has my proliferation gone?!

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Steff
Steff's picture
Where has my proliferation gone?!

Hi, I'm really hoping someone can help with this - or at least offer some suggestions. I'm tearing my hair out here.
I'm culturing mouse lymphocytes for 5 days in HL-1 media plus 100ug/ml pen/strep, 1% L-glut and 2.5% beta mercaptoethanol, with 25ug/ml peptide antigen. On the 5th day I add 1uCi/well H3 and then, in theory, measure the proliferation. Only....nothing. No proliferation.
This started a couple of months ago - the proliferation went from decent counts of about 10000cpm for positive wells to 0. The positive control of conA is still giving cpm's of 100000, but everything else is missing. And everytime I've repeated it, I get the same results, even on peptides I KNOW should give positive responses. I've checked the media (works with other cells), the incubator is fine (all the other cells in there are happy and healthy and CO2 levels are normal), I've decreased the antibiotic concentration, I've used new batches of everything (media, pen/strep, L-Glut, thymidine, antigen etc), I've used cells from different mouse strains, I don't have any contamination in the plates, I've even had someone stand and watch me to check I'm not going crazy!
And here's where it gets even weirder - when we check with Elispots, the cells are definately kicking out IFN-y at expe3cted levels in response to the antigens, so they're alive and responding, they just refuse to proliferate.
Has anyone had a similar problem or even heard of something similar? Am I overlooking something obvious, Or have I discovered a selective way of stopping cells dead in their tracks?

marcus muench
marcus muench's picture
Is there any chance that you

Is there any chance that you have an increase in cell death leading to increased DNA in the cultures, which could act as a cold chase to your hot 3H labeled thymidine?

Also check your timing. You may be missing peak proliferation. Check earlier and maybe later time points. In other words titrate time and then try titrating the peptide. It is hard to go wrong with ConA, but Ag-specific responses can be more tricky. You may have just gotten lucky in your first experiments. Any chance your peptide solution has gone off?

samm
samm's picture
Carson O Genic wrote:Is there

Carson O Genic wrote:

Is there any chance that you have an increase in cell death leading to increased DNA in the cultures, which could act as a cold chase to your hot 3H labeled thymidine?

Also check your timing. You may be missing peak proliferation. Check earlier and maybe later time points. In other words titrate time and then try titrating the peptide. It is hard to go wrong with ConA, but Ag-specific responses can be more tricky. You may have just gotten lucky in your first experiments. Any chance your peptide solution has gone off?

I totally agree about the time points - I've never gone over 72 h (+16 h TdT pulse) for ova-APC (B6 or Balb/c) or ova peptide (98% DO11 T cell, no APC; also with APC). The peak for Con A is actually sooner. You seem to be following the protocol for human T cells,which are generally cultured longer. Adding TdT at the tail end of your proliferation cycle will give very low counts: also, Con A may be able to sustain both cytokine production and proliferation, but peptides (esp in a APC low/free T:T culture) may not be able to get to the apex of the activation pyramid.

The 'competition' by cold DNA is somewhat unlikely but possible - DNA fragments from apoptotic cells have to undergo two additional rounds of processing, while TdT is immediately available. Have you tried doing a cell cycle/hypodiploid staining (PI) with these cells?

samm
samm's picture
Also, try reducing b-ME

Also, try reducing b-ME amounts to 10-50 uM. b-ME does improve primary cultures, but too much can be bad.

tcells4ever
tcells4ever's picture
Hi,

Hi,
I just wondered if you found out what the problem was with the proliferation assays.  I'm having similar problems and have also tried troubleshooting everything I can think of without fixing it.  If you found out what it takes to get around this, could you let me know?