I am stimulating murine macrophages (RAW 2264.7 cells) with doses of LPS ranging from 0.1 ug/ml to 100 ug/ml (for 24 h) but not seeing any IL-6 production at all (as measured with an ELISA). I ran these experiments back in 2012 and saw tons of IL-6 at all these LPS concentrations (RAW cells treated with all these concentrations of LPS produced over 1000 pg/ml of IL-6). I'm repeating these experiments and not seeing any IL-6 production. I wonder if it has to do with the microcentrifuge tubes I am storing the LPS stock in -20 in (the stock is 2 mg/ml and I make serial dilutions for the treatment concentrations). I vaguely remember using Lo-bind microcentrifuge tubes to store my LPS stock back in 2012, which I am not doing now. But I don't think using normal microcentrifuge tubes would be causing this lack of effect! Sigma is trying to sell me sialized glass vials to store my LPS stock but they are very expensive. Does anyone use these? Can anyone think of why these experiments are not working?
Hi Christina I have used Sigma LPS and have had no problems (so far!).
As LPS is negatively charged I would use DNA LoBind tubes (as DNA is also negatively charged).
Obviously any reagent has a finite storage time and I assume you are not using LPS that has been stored at -20 for longer than one year.
Another possibility may be your cells of course. Are you using the same passage/cPD cells as in your original experiment?
A second possibilty is your IL-6 detection method (although, if i.e. Elisa I assume you are using a positive control/standard curve to eliminate this possibility).
To check it is not your cells I suggest you purchase more LPS (it is fairly cheep) and perform a stimulation on the cells with FRESHLY prepared LPS (i.e. no storage). If you get an IL-6 response you can repeat this with -20 LPS (same stock) to see if storage is causing the problem. If it is this, worst comes to worst, you can buy small amounts of LPS each time for your experiments (probably work out cheaper than buying silanised vials).
Personally as you are performing a titre and not seeing ANY IL-6 - even at the higher dose I suspect it may in fact be the cells....(or another factor).
I have always stored LPS in normal cryovials, not even the lo-binding ones.
First thing is I would look at the morophology of your cells under LPS activation, they should demonstarte a clear difference from the unstimulated populations (especially at the highest concentration). If they look activated, and you see no IL-6 in your ELISA, then I would assume it's the ELISA you using.
If the cell do not "appear" activated, start backtracking as Gary suggested. Prepare a fresh LPS stock. And if that still doesn't work, you might need to reorder your cell line.
Good luck!
I agree with the above answers. LPS stored in the most basic tubes should be fine. Fancy ones are totally unnecessary and a waste of money.
You could have an issue with your particular LPS stock - this is easy enough to test by treating your favorite control cells that *always* respond well to LPS and seeing what happens.
My honest guess is that the cells are your issue. If so, here are important troubleshooting questions:
1) How long have these been in the freezer?
2) How many times have they been passaged?
Even the most stable of cell lines starts acting very differently after a few dozen passages. Macrophages may change considerably sooner as they naturally want to differentiate and adapt to different environments.
3) Have you tested your cells for mycoplasma contamination? It is almost impossible to detect by eye and very common in laboratory cell cultures, is unaffected by most antibiotics, yet messes with TLR activation and affects immune responses such as IL-6 secretion.
4) Are you growing your cells in media containing antibiotics? I have found that antibiotics, even in standard concentration, seriously affect the growth and immune responses of cells in culture. Even if you regularly passage your cells using antibiotics, please take them off of antibiotics when you're plating them for experiments for the sake of reproducibility.
Finally, I would always highly recommend getting a new stock of cells from ATCC or another well-tested commercial source if you're trying to repeat important experiments. A dismayingly large percentage of research is done on old/contaminated/misidentified cells, and we all fall into that trap far too much. 99 out of 100 times, starting fresh will save you a ton of time and money you'll spend troubleshooting and having difficulty in reproducing experiments.
Good luck!
I have been taught to vortex LPS vigorously for at least 1 min before pipetting each time. That way it will disperse into smaller chunks/micelles and it will not stick as much to the tubes.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue