I evaluated the intracellular bacteria (mycobacterium spp) killing effect of bone marrow-derived macrophages(BMDM-adherent cell) through Gentamicin protection assay.
In the procedure of Gentamicin protection assay, cells are washed out by D-PBS after 1h post infection. Then cells which were infected with intracelluar bacteria are incubated with fresh medium containing gentamicin for 48h, 72h. Next, at the indicated time point, cells are washed out by D-PBS and lysed by Triton-X.
In this process, dead cells(Dead cells containg live bacteria may also be present) would be removed through D-PBS wasing because dead cells are floated from the plate. Also, Bacteria which were released from dead cells would be killed by gentamicin in the medium and removed by the PBS wasing process.
For this reason, I think that cell death is a very important control variable in performing the gentamicin protection assay. However, many papers (containing below two paper) do not evaluate cell death as a control variable.
So i think that, when evaluating the bacterial killing ability of a specific host protein, the evaluation through the GENTAMICIN PROTECTION ASSAY can demonstrate the direct killing ability of the protein, but the effect of cell death should be excluded.
I hope to hear several opinion from experts.
Thank you
Immunity. 2019. PMID: 30683619
Cellular Microbilogy. 2011. PMID: 21790937
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