PKhLC3 plasmid is to observe the autophagic dynamics. The EGFP-Phluorin will disappear if the autophagosome fuses with the lysosome due to the acidic pH of the lysosome. So we expect to see only red puncta when autophagy is going on. We are doing some experiments with Hela cells where we did a transient transfection with the PkhLC3 plasmid. After transfection, we serum-starved the cells for 18hr to induce autophagy. We did see green-red puncta in both our control and knockdown cells. But unfortunately, we did not see any red puncta at all even in the control! Does anyone have a similar problem with plasmid? We would appreciate any suggestion.

Thanks,

Samina.