cell line nonresponsiveness - is it a time to change my cell line???? (May/21/2006 )
hi all ! iam working on Toll like receptor signaling and cytokine profile studies in J774 mouse macrophage cell line. so far i have done western for MAP kinase study(ERK1/2 and p38) and ELISA for interleukin study (IL-10 and IL-12p70). i didnot get any response when i triggered the cells with TLR ligand. i have done time dependent studies and concentration dependent studies but all failed. ligand is working fine as the same aliqote used by another person (working on THP human macrophage cell line). this ligand can trigger both the cell line.
doubling time for J774 is mentioned as 36hrs by ATCC but my cell line takes just 12-18hrs for doubling. coming to my question can i say that my cell line has reached a state of non responsiveness? iam using 4500mg/L glucose but recommended is 1000mg/L cani say that glucose is the culprit? how about using primary cells?
iam rather new to this field.hope someone can help me out !!!!!
I know macrophages tend to express alot of different TLRs but is it worth checking for your particular TLR expression by flow cytometry. If you're worried about the growth of the cells you have I'd advise taking a fresh aliquot from nitrogen. Is anybody else in your lab working with that cell line ? If so you could check with them about growth rates and culture conditions. Do you serum starve the cells prior to stimulation to quiesce them?
The Raw264.7 cell line is a commonly used mouse macrophage cell line in TLR/LPS studies maybe you could use that instead or the human THP-1 line.
All the best,
Ceri
hi ceri thanks a lot for the reply ! i think you are right . either i have to take out a new lot ( but our cryopreserved stock is from those non responsive cells) or change my cell line (i think its the best option. mouse RAW cells are more responsive compared to J774, thats what i heard from others. unfortunately no body else is working on signaling in my lab. iam thinking of planning my expts on primary cells. poor mice..... i have to sacrifice so many of them.........
also ceri thanks for the suggestion of serum deprivation. this time i will keep this in mind.
have a nice day
