Murine Macrophage - Il-12 and Il-10 Expression? - (Jan/08/2006 )
I've been trying to look at mRNA expression of IL-12, Il-10, TGF-Beta, Il-1b, and IL-1ra...IL-12 and IL-10 do not seem to be expressed in the murine RAW 264.7 cell line I'm using when using RT-PCR (and also pushing them towards expressing it using a variety of activation statres).
Would the ELISA method be the quickest and best way to determine their expression considering IL-12 and IL-10 expressoin is probably in the 10-100 pg/ml range? RT-PCR is more in the ng/ml range which I don't think is sensitive enough for IL-10 and Il-12 expression.
Also, just a general question..when exposing RAw264.7 macrophages to LPS/IFN-gamma...I waited 4 hours before extracting RNA..should I wait longer??
Thanks for any responses or suggestions you guys can offer.
mmm...the application is different...but we use all sorts of things to stimulate our epithelial cells...LPS, IL-1beta, LTA, other stuff...when looking for expression changes with innate immunity factors, we always start with a timecourse. In my experience, most stimulants in a reasonable concentration will show expression change in 90' to 5 hours, but there are always some that take longer. What are the signalling pathways involved? for each cytokine, there is a predictable lag for expression....this was in another thread not too long ago; I believe it was AussieUSA who gave some tips on how long it takes for cytokine expression?
anyways, when we are looking at a new stimulant and we're not sure, we start with a rough time gradient, because sometimes you might miss the peak entirely, if the mRNA turns over into protein and undergoes degradation rapidly (for example, sometimes NFkB signalling has multiple peaks in the cell, based on the signalling involved; you will see an early spike and a late spike if you are looking at expression driven by this pathway)
Thanks for the suggestion. I had seen that post, but he didn't give a definitive time frame on those cytokines' expression..I tried post-4 hour activation, I'll try 8 hours this time around.
BTW, when you attempt your own activation in your cells do you use media without FBS?
we don't look at macrophages; we look at epithelial cells. we look at cytokine and defensin induction. the only time we ever use FBS is immediately after trypsinization, then we rinse it off...there are too many immune factors and such present in FBS; it would throw off all the numbers
I don't know if I have been helpful, since the cells are so different. we have seen some groups who look at cytokine induction go as long as 2 or 3 days...we think it is difficult to narrow down the actual cause of the induction, if you go that long; there is so much redundancy and feedback stimulation by other molecules...we try to always keep our assays within 24 hours, usually much less time
what do other people in your field use, as a reasonable time frame?
we use primary human and mouse macrophages and look at IL-10, IL-12, TNF expression after LPS stimulation. TNF is the fastest of these three and you should be able to pick it up after 1 or 2 hours (fold inductions up to 800-1000), IL-12 and IL-10 are a lot slower, you should be fine after 8 hours of stimulation for these cytokines. Fold inductions are lower for these two, usually between 5-20 fold depending on the concentration of LPS you use.
As for protein measurements, we wait for 24 hours and perform ELISA on the supernatants. We are able to pick up all three cytokines at that time point