I've been infected bone marrow derived macrophages for years now with various strains of Francisella tularensis but the cells will not become infected anymore.
I use bone marrow derived macrophages, cultured with L929 media (tried three different batches of L929 media from different frozen stocks of cells, my own stocks from 2012 and 2014 batches). I've used different sets of culturing reagents, however when I do this experiment with J774 cells instead of bone marrow derived macs, I have no issues - bacteria replicate as normal.
My bone marrow derived macropahges are indeed F4/80 high, CD11b high, CD45 high, CD11c -. They are healthy looking at naive and infected looking when infected. When infected and treated with LPS by day 2 they look rounded and activated, but healthy looking. Yet when I plate the cells at day 3 I see no colonies. If I use J774 cells, the bacteria grow as normal so my saponin for cell lysis and my plates are functioning correctly.
I infect my macs for 2 hours then wash, then treat with genta, then wash thoroughly and plate the cells to see if bacteria are being taken up by my bone marrow macs. I see plenty of bacteria as normal at this time. Also my innoculum counts are good. But somehow over the three days in culture all bacteria disappear. So strange, even LPS treatment normally allows for some bacteria to survive.
I'm going to try adding L929 media to J774 cells to see if somehow all my L929 stocks are somehow delivering something that prevents my bacterial replication. Would mycoplasma do this? or some virus? Or perhaps my Balb/c mice are infected from the start wtih something? What's odd is that I made my L929 cell stocks in 2012 and 2014, and they worked fine before, so I can't conceive of how they would be to blame now. But I suppose it could be some plastic ware too? Perhaps the large filters I use to filter the culture. I feel as though I'm grasping at straws...so frustrating.
I appreciate any input anyone can offer.
The simplest way to find out was it effect on the macrophages by viruses - is to determine their level of expression of TLR 3. If the presence of the virus it was to be an increase in expression of TLR 3.
Dear Felicity, happy to hear from you! I would check the presence of antibiotics in media.
Dear Felicity, your macs could be activated before FT infection...? but why?...or try to prepare your FT stock again, resuspend in other media or check properly the growth...try to check other MOI.. or check if L929 stock has not been thawed repeteadly ......and what about cytokines, control x early immune response? Of course co-infection may be.on the programme....
Dear Felicity all the previous answers are reasonable. I would suspect that the cells are contaminated, quite easy if they have several passages. However, as soon as you get a new batch growing test for fungus infection.
regards,
Thanks everyone for the suggestions. I really appreciate you're taking the time to write.
I've replaced the media with new batches a few times, and all works well with the cell line J774 - so I don't think there could be antibiotics in the media. This experiment has been failing over 6 times now, I'm just in pure troubleshooting mode.
The L929 stock I took from was my original batch in 2012 (always worked in the past). However I am going to try and differentiate bone marrow cells with purified MCSF to see if the L929 cells are somehow faulty. Or maybe it's the filters I use to filter the media before adding it to my bone marrow cells? Seems such a long shot. But i suppose if the filters (regular disposable filter units) had an endotoxin of some kind in it?
It is reasonable to think that my mac's are activated to start with, but I would think that even with activated mac's they don't typically kill all the bacteria? But a possibility I have to consider. If the L929 cell supernatents are activating them, I should know in a few weeks when I work with the MCSF differentiated mac's.
It's a long shot, but it might be that the balb/c mice in the animal facility are infected with something? I'm not sure what kind of virus would cause this, but I'm going to send off some mouse samples for testing at Charles River.
I have no experience with what a hepatitis virus or other sort of virus might do to the mice, whether the virus would even make it to the bone marrow.
In the past we had something similar, but our problem was FT stock ( plate from plate) which we have to pass through mice to overcome the problem. we are working with human monocytes and macrophages.
The simplest way to find out was it effect on the macrophages by viruses - is to determine their level of expression of TLR 3. If the presence of the virus it was to be an increase in expression of TLR 3.
Ok, thanks Andrew. that's a good idea. I can easily look for that in my next experiment.
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