I am trying to set up an in vitro experiment where I co-culture peritoneal macrophages and T cells together in the presence of HMPV infection. After incubating the macrophages and T cells together, I harvest the cells and run flow cytometry looking for T cell activation via Ki67 and CD44 markers.
I have repeated this experiment three times, adjusting the conditions between the three replicates and each time I see that the uninfected macrophages and T cells are more activated than the infected groups (i.e expressing more KI67 and CD44).
I tried removing mCSF from the media after setting up the co-culture since I thought this might be activating the macrophages. I also tried various lengths of times for the co-culture ranging from 48hrs-4 days. I added IL-2 to the media to help promote T cell survival. In addition, I use a 96 well u-bottom plate and added the macrophages: T cells in a 1:10 ratio to ensure adequate T cell activation.
Please let me know if you have any suggestions for what I should try next. Thank you!
Dear Olivia,
I am no expert concerning T cell culturing, so here is a T cell expert needed ;) We incubate our peritoneal macrophages in DMEM + 10% FCS (heat-inactivated) without anything in addition. They are not like BMDM, which need the M-CSF. Since you did this already this is a good step. Perhaps you can give me some intel of your macrophage preparation and I will try to help you. We worked with peritoneal macrophages for years and perhaps we can find a solution. Moreover, I have never heard of KI67 (which is involved in cell division) or CD44 (an adhesion molecule) as competent markers for macrophage activation. Perhaps these parameters may be also the problem.
All the best,
Marc
Marc Herb :
Hi Marc,
Thank you for the feedback! I harvested the peritoneal macrophages using PBS and an 18G needle. I sac'd the mouse, sprayed the abdomen with 70% EtOH, and made a small cut into the skin to expose the peritoneum. Using the 18G needle, I flushed PBS into the peritoneum. I repeated this about 4-5 times and got around 7-8mL of PBS back. I spun this down, performed RBC lysis, and counted the cells before plating on the 96 well-u-bottom plate. In previous experiments, I have run flow cytometry on the harvested cells directly ex vivo and found that this approach yields ~70% purity of CD45+ F4/80+ cells.
I isolated T cells from the lung using Ficoll and also counted the cells before plating with the peritoneal macrophages on the 96 well-u-bottom plate. The CD44 and Ki67 markers were used to assess T cell activation not peritoneal macrophage activation. To assess peritoneal macrophage activation I used CD80 and CD86.
For this particular experiment, I also was looking at PDL1 expression on the peritoneal macrophages since our lab has previously shown that in the presence of HMPV infection, macrophages upregulate the inhibitory ligand, PDL1. My hypothesis for this experiment was that the macrophages are impairing T cell function by upregulating PDL1 expression, which is why I was looking for PDL1, CD44, and Ki67 expression.
Let me know if I can provide any further details.
Thank you for your help,
-Olivia
Dear Olivia,
your protocol for peritoneal macrophage isolation sounds good to me. I am a little bit curious about the purity. Before magnetic cell sorting we only get 40% of macrophages from the peritoneum (gated to CD11b and F4/80).
The other cells are mainly B cells and a few neutrophils. So when you plate your whole lavage without seperating the peritoneal macrophages, you will get a co-culture of your T cells with a mix of cells but for sure not only with peritoneal macrophages.
B cells and Neutrophils might not only influence your macrophages, but also your T cells. If not with secreted cytokines or other molecules, just because they will die over night and release DAMPS. So basically you confront your T cells with a undefined mixture of cells and molecules released into the medium.
I think a pure culture of peritoneal macrophages will solve your problem. And then you can infect them and you will only have the PDL1 upregulation as factor that might influence your T cells.
I hope that helps you. For any more questions please feel free to ask any time :)
All the best and stay healthy,
Marc
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