CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit helps to monitor Autophagy in live cells by Flow Cytometry, fluorescence microscopy and microplate reader.	MLO-Y4 cells to stain with Cyto-ID to visualize autophagosome following Dexamethasone treatment	Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Upstream tips
This kit helps to monitor Autophagy in live cells by Flow Cytometry, fluorescence microscopy and microplate reader.
Protocol tips
MLO-Y4 cells to stain with Cyto-ID to visualize autophagosome following Dexamethasone treatment
Downstream tips
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Publication protocol

MLO-Y4 cells cultured on glass coverslips and calvarial primary osteocytes ex vivo cultured in 96-wells plates were fixed for 15mins and and 2hrs at RT with 4% PFA respectively. Cells were then permeabilized for 5 minutes with 0.1% Triton X-100 in PBS, and non-specific antibody binding was blocked by 3% BSA-PBS for 30mins. MLO-Y4 cells and primary calvarial osteocytes were incubated with primary antibody diluted in 0.2% BSA-PBS for 2hrs at RT or overnight at 4°C respectively. Following incubation cells were washed extensively and bound primary antibodies were reacted with fluorescence conjugated secondary antibody for 1hr at RT, and nuclei were stained with Hoechst 33258 dye for 15mins at RT. Coverslips were then mounted in ProLong Diamond Anti-fade medium (Invitrogen, USA). Images were acquired on a Nikon A1 confocal microscope equipped with 60X (oil immersion) lens, and analyzed using ImageJ software. For quantification of the number and length of dendritic processes and the number of Cx43 fluorescence dots and endogenous LC3GFP punctual of each osteocyte, the channels were extracted and converted to greyscale images and analyzed using ImageJ software.

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