I tried to see the autophagy formation in the Rapamycin treated cells by using autophagy dye.Did any one tried by measuring fluorescence of autophagy dye added to the cells after induction?
Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles
Dear Nageswar,
You can monitored autophagy induction by using acidine orange dye through fluorescence microscope.
In my experience the best kit is Cyto-ID that selectively labels autophagic vacuoles.
Link to data sheet is
http://static.enzolifesciences.com/fileadmin/files/manual/ENZ-51031_insert.pdf
Either MDC or Cyto-ID can be used but you need to be very careful about how to interpret the findings. At least another assay need to be used, especially the measurement of autophagic flux.
Hello,
Regarding autophagy dye, you can use MDC (Monodansylcadaverine), a potent marker of autophagy (Biederbick et al.1995). Futhermore, if you like to observe autophagic vacuole and fusion with lysosome you can do double fluoresence staining with two primary antibodies [anti-MAP–LC3B (sc-28266) and anti-LAMP (sc-
65236)] to detect vacuoles and matured vacuoles. YOu can also do the fluorescence study with only anti-MAP–LC3B (sc-28266).
Wish you the best
Here attached a "consesual recepice" about how measuring autophagy.
Best regards
Robert
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