I am planning to carry out gentamicin protection assay to study the internalization of E.coli in macrophages and dendritic cells. I want to specifically look at the degradation of E.coli in lysosomes. Does anyone have an idea that how long does it take for E.coli to reach lysosomes in RAW cells and primary macrophages and dendritic cells from mice?
Hi,
I am not sure about dendritic cells, but primary macrophages such as peritoneal macrophages or bone marrow-derived macrophages need only a few minutes to take up E. coli or L. monocytogenes. We analyzed uptake kinetics of different bacteria by IF microscopy and by FACS and we observe that already 15 min after infection, about 80 % of L.m. or E.coli are intracellular.
To be sure you can add your gentamycin to the cells 1 h after infection. At this time point almost all bacteria should be internalized. I would suggest to use an MOI between 0.5 - 2, not higher.
We analyze the phago-lysosomal degradation by two different methods, either by IF microscopy (co-localization of lysosomal fluid phase markers with the internalized bacteria) or by analysis of acid hydrolase activity in a plate reader (by coating the bacteria with probes that become fluorescent upon cleavage by acid hydrolases). Here we observe that 15 min after infection about 60 % of internalized bacteria co-localize with lysosomal fluid phase markers. The fluorescence signal in the plate reader, which represents acid hydrolase activity, starts to increase about 30 min after infection and reaches a plateau about 2-3 h after infection.
To get an idea, whether your E.coli have been degraded, you can lyse your cells and plate the lysates on ager plates at different time points after infection to detect living bacteria by colony formation (CFU assay). 5 h after infection, there should be only a few colonies left in comparison to your starting point. 24 h after infection, I would expect to have no colony formation at all, since the E.coli should be completely degraded at this time point.
For further details and protocols, you can have a look at our recent studies, where we used the above described methods:
The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes. Cell Host & Microbe, (2018)
DOI: 10.1016/j.chom.2018.01.018
Macrophages target Listeria monocytogenes by two discrete non-canonical autophagy pathways.
Autophagy, (2021)
DOI: 10.1080/15548627.2021.1969765
Kind regards,
Alexander Gluschko
Dear Dr. Alexander Gluschko
Thank you very much for the protocol. This has indeed been very helpful.
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