Splenocyte Proliferation Assay

[luisfigo]

I am facing a problem with my splenocyte prolifertion assay. I am harvesting mouse splenocytes with standard protocols and then using 0.1 million cells per well in a 5 day protocol. I check my cell viability by trypan blue after harvesting and they are 95 % live cells. After 5 days i add 0.5 micro curie of thymidine per well and harvest after 18-24 hours. However after harvesting i do not get any counts even in the positive control like Con A or PHA. The wells have basal counts similar to negative wells or as the wells where i have added the antigen for stimulation. The same positive control has been used with human T cells and DCs and is working fine. I am facing a problem only with mouse splenocytes or lymphocytes.
I use RPMI with 10 % FCS and beta mercaptoethanol.

Ive also used media without mercaptoethanol but its the same thing.The media colour is still pink indicating the pH is ok and has not turned acidic.

I would appreciate if anyone can help me finding a solution to my problem. I am really stuck here and have repeated at least four to five times.

[darkwng]

may not be any help, as I have only performed one of these assays (and it was pretty dodgy- I'm only an honours student!!) But, I did get a detectable proliferative response. (my splenocytes were from BALB/c mice).

The media formulation I used was RPMI 500 mL, 2-mercaptoethanol 0.1mM, HEPES 10 mM, Non-essential amino acids 1%, Sodium pyruvate 0.1 mM, L-glutamine 0.02 mM, FCS 10% (and media antibiotics).
I didn't actually use the thymidine method for measuring proliferation though, I used an MTT tetrazolium salt colourimetric assay. This involves adding MTT to each well and incubating for 2 hours, then adding an extraction buffer, allowing colour to develop overnight and reading the OD using an ELISA plate reader.
You probably won't want to change your thymidine method for this one though?? I'm not sure but I think your way is more sensitive?? Anyways, let me know if you want to give it a go and I could give you a protocol.

[arpineee]

Hi,

Can you please send me your protocol of MTT assay, which you are doing on BALB/c mice splenocytes. I have tried twice, I cant get anything. It will help me very much
Thank you

[patriciapan]

Hi, I am also having problems on splenocyte proliferation assay. I am having problems with my splenocyte proliferation assay. Do you use flat bottom 96 well plates? Treated or non-treated? I incubated my spleen cells for 68 hours then when i checked using mtt, i think majority of the cells are dead coz there is not much crystal formation. And I also tried using cona as a positive control, and there wasn't much proliferative response.
Thank you. Your help would be kindly appreciated.

Regards,
Patricia