Dansylcadaverine

Protocol tips

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	Incubate with 50 μM of MDC in PBS 1x at 37 °C for 15 min

Protocol tips
Incubate with 50 μM of MDC in PBS 1x at 37 °C for 15 min

Publication protocol

Autofagy was analyzed by flow cytometry by using monodansylcadaverine (MDC)(Sigma-Aldrich, Saint Louis, Missouri, Stati Uniti) staining. MDC is an auto-florescent agent used as a selective marker for autophagic vacuoles (AVOs) and especially autolysosomes. U87MG cells were plated in 6-multiwell plates at the density of 2 ×105 cells/well. After 72 h of treatment cells were incubated with 50 μM of MDC in PBS 1x at 37 °C for 15 min. After the incubation the cells were washed twice in PBS 1×, trypsinized and resuspended in 500 μl of PBS 1×. Analysis was performed by flow cytometric (BD Accuri™ C6). The fluorescent emissions were collected through FL1 channel. For the quantitative evaluation of MDC, BD Accuri™ C6 software (Becton Dickinson) was used to calculate MFIs. The MFIs were calculated by the formula (MFI treated/MFI control), where MFI treated is the fluorescence intensity of cells treated with the various compounds and MFI control is the fluorescence intensity of untreated and unstained cells. For each sample, 20,000 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.

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