Hie
I am working on a peptide which is inducing autophagy in THP1 derived macrophages and thus clearing mycobacterial infection. I found upon peptide treatment, the M.bovis infected macrophages are apperaing multi vacuolated which is not in the case of infected control cells . Adding wortmannin along with the peptide in the cells is rescuing the multi vacuolated apperance of the infected macrophages. In the image attached i have performed LC3 staining. The peptide is not inducing cell death as i have checked it with annexin V/fitc flow cytometry assay. What could be the possible reason to this multi vacuolated nature?
Additional information about culture conditions and a phase contrast image could help in interpretation of the observations.
Hello Kavita.
It seems that the infection effects are under thresh concentrations to cause autophagy. And the infection duration and the source of the microbes, their different specificities to macrophage and heated or live are the other factors to be considered before the threshold concentration. And, too.
I suggest that determine IC50 with consistent infection strength to test intracellular ROS and cell death first. Based on the determined IC50 concentration of infection and duration, set several levels of infection strength around the IC50 levels.
This determination will tell why you did not see autophagy, but see only vacuolar structures.
Best wishes.
Thanks for the reply Dr. Lee
I have used MOI 1:10 for the infection and have checked mROS using Mitosox based flow cytometry at same peptide concentration with and without infection in THP1 derived macrophages. The peptide have shown slight increase in mROS level (6-7%) in infected macrophages whereas same peptide is showing slight reduction (7-8%) of mROS levels in uninfected macrophages. The peptide do not exert any effect on proinflammatory cytokine levels.
Hi, Kavita.
Although you did mitROS at the concentration of 1/10, demonstrating only 6~7%, measurement of intracellular ROS accumulation would be more appropriate since the MAP1LC3 are associated with whole cytoplasmic components. The 6~7% increase would not be enough to cause autophagy, thus explaining your results in the figures.
Using DCF-DA for the measurement you could go higher concentrations of MOI infections that bring about IC50 (inhibition of cellular activities) in antioxidant activities of the cells. That has to be determined first, and then sets several concentrations around it, making sure that your cells are under being damaged. From the determined concentration, you may get autophagy.
If DCF-DA and spectrofulorimetry measurement are not accessible at present, do the same above, followed by simple propidium iodide/Hochst33322 dual staining for cell death, determining LD50. Do similar evaluations to find an appropriate concentration for autophagy experiments.
Best of luck
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