Morning
I currently have the dilemma of detecting M.tuberculosis in a BMDM assay looking at the efficacy of drugs in killing intracellular M.tuberculosis.
Ideally, it would be beneficial if I could detect presence of M.tuberculosis without needing to lyse the BMDMs, but I understand that lysing the BMDMs to detect the M.tuberculosis expressing GFP is necessary. However, after day 6, it is quite difficult to detect the GFP using only a fluorometeric device. I have read up on the subject and find that most laboratories are now looking at microscopy to detect presence of GFP expressing M.tuberculosis (E.g. http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1003946)
Does anyone have or know of any protocols used within their laboratory which uses GFP to detect TB presence in BMDMs? Or should the BMDMs be lysed to release intracellular bacteria how would you separate the TB from the solution containing drug, cellular contents etc.
Thanks
Dear Michael, I have no experience and no idea about using GFP to detect TB, nor working with macrophages so excuse me if what I 'm about to write is stupid.
Some time ago was described that Mycobacteria is autofluorescent (emitting in cyan when excited with UV). Maybe you could take advantage of this?
I don't know what molecule is absorbing UV to emit in cyan, so I can't tell you if it will be differences between alive and dead bacteria and I don't know if this fluorescence would be detectable without lysing the macrophages either.
Just some crazy ideas; hope this helps ^^'
counting of GFP microscopically is not always accurate as it is not giving the accurate number of the bacteria/ cell, besides the extracellular located bacteria is difficult to be excluded especially some mycobacteria species have ability to transfer between the adjacent cells this will affect the counting as well, and we must not forget that the CFU is not reflect the bacterial number because some bacteria not give CFU on agar.
i was working on M. avium and was really difficult to differentiate between alive and dead bacteria microscopically without counting CFU and comparing with fluorescence however, this was rationally as well.
Dear Michael
You may find the following publications useful as a start.
Bettencourt, Paulo, et al. "Application of confocal microscopy for quantification of intracellular mycobacteria in macrophages." Microscopy: Science, Technology, Applications and Education 614 (2010).
Lowe DM, Bangani N, Mehta MR, et al. A novel assay of antimycobacterial activity and phagocytosis by human neutrophils. Tuberculosis (Edinburgh, Scotland). 2013;93(2):167-178. doi:10.1016/j.tube.2012.11.014.
Jordao, Luisa, et al. "On the killing of mycobacteria by macrophages." Cellular microbiology 10.2 (2008): 529-548.
Kumar, Shashi Kant, Padam Singh, and Sudhir Sinha. "Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation." Open biology 5.12 (2015): 150171.
These use fluorescent mycobacteria (GFP expressing or stained) to quantify intracellular mycobacteria by microscopy. I have some experience applying this to flow cytometry, and I can confirm that you can detect intracellular GFP-expressing mycobacteria post-infection. If you think I can help with anything further, do let me know.
Best,
Ashley
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