I have been trying to change lysosomal pH using ammonium chloride or chloroquine for a while now but I haven't had much luck. I am measuring the increase in pH using ratiometric analysis where I use pHrodo green dextran as my pH sensitive dye, and dextran 647 as my pH insensitive dye.

For my treatment, I have tried creating a 10 mM ammonium chloride solution in HBSS (diluted from 10X HBSS without bicarbonate or phenol red) or DMEM and washing the cells with it for maximum 30 minutes. After ratiometric analysis, I see no difference between control and treatment group. Similarly, I treat cells with 60 uM chloroquine for 30 minutes and observe no effects on pH (both using HBSS or DMEM).

I have used Concanamycin A before (which inhibits V-ATPase directly) and that seems to work perfectly fine. However, it also affects downstream proteins like mTORC1 which I do not want.

Any help with this would be greatly appreciated!