CYTO-ID® Autophagy detection kit

Protocol tips

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	Lysotracker Red (Invitrogen, San Diego, CA, USA) was used to stain lysosomes and autophagolysosomes. PC-12 cells were incubated with PAMAM dendrimers G5 for a series of times, then Cyto-ID Green Dye, LysoTracker Red, and Hoechst 33342 were used to stain cells at 37 oC for 20 min. After that, cells were washed in RPMI-1640 medium and observed by confocal microscopy immediately. All the relative fluorescence intensity was measure by inverted confocal microscope software (Carl Zeiss LSM710, Carl Zeiss, Germany).

Protocol tips

Lysotracker Red (Invitrogen, San Diego, CA, USA) was used to stain lysosomes and autophagolysosomes. PC-12 cells were incubated with PAMAM dendrimers G5 for a series of times, then Cyto-ID Green Dye, LysoTracker Red, and Hoechst 33342 were used to stain cells at 37 oC for 20 min. After that, cells were washed in RPMI-1640 medium and observed by confocal microscopy immediately. All the relative fluorescence intensity was measure by inverted confocal microscope software (Carl Zeiss LSM710, Carl Zeiss, Germany).

Publication protocol

1×105 cells were cultured in dishes with glass bottom for 12 h, and then treated with a series concentrations of PAMAM dendrimers G5 for different times. Cells were disposed with Cyto-ID Autophagy Detection Kit (ENZO Life Science, Farmingdale, NY, USA) and MitoSox Red mitochondrial superoxide indicator (Eugene, Oregon, USA) following the manufacturer's instruction 30. The images were observed and overlaid by an inverted confocal microscope (Carl Zeiss LSM710, Carl Zeiss, Germany). Lysotracker Red (Invitrogen, San Diego, CA, USA) was used to stain lysosomes and autophagolysosomes. PC-12 cells were incubated with PAMAM dendrimers G5 for a series of times, then Cyto-ID Green Dye, LysoTracker Red, and Hoechst 33342 were used to stain cells at 37 oC for 20 min. After that, cells were washed in RPMI-1640 medium and observed by confocal microscopy immediately. All the relative fluorescence intensity was measure by inverted confocal microscope software (Carl Zeiss LSM710, Carl Zeiss, Germany).

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Manufacturer protocol

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