Autophagy detection kit

Chloroquine diphosphate was purchased from Sigma Aldrich (St. Louis, USA). The primary rabbit monoclonal antibodies against LC3-I, LC3-II, ATG3, ATG5, ATG6, ATG7 and β-actin were obtained from Cell Signaling (Danvers, MA, USA). The goat anti-mouse/rabbit IgG secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The FITC-Active Caspase-3 Apoptosis Test was obtained from BD Pharmingen (San Diego, USA). For monitoring of autophagy in living cells by immunocytology and flow cytometry the Autophagy Detection Kit (Abcam, Cambridge, UK) was used. Hoechst 33258 was obtained from Sigma, Rotitest Vital from Roth (Karlsruhe, Germany). TUNEL assay was obtained from Roche (Mannheim, Germany). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA) and siRNA oligonucleotides against ATG3, ATG5, ATG6, ATG7 and scrambled siRNA from Dharmacon, Lafayette, USA. Their sequences have been published before [28 (link)].

Autophagic flux measurements were performed using an Autophagy Detection Kit (ab139484; Abcam, UK) according to the manufacturer’s protocol. Cells were seeded into 24-well plates at a density of 10,000 cells/well. At the end of the treatment, autophagic vacuoles were stained according to the manufacturer’s instructions before being imaged under fluorescence microscopy (ImageXpress Micro 4 High-Content Imaging System, San Jose, CA, USA) [54 (link)]. For flow cytometric analysis, after treatment with BJA, cells were collected and then washed with 1× assay buffer. After that, autophagosomes and autolysosomes were stained with a green fluorescence dye for 30 min of incubation and then analyzed quantitatively by flow cytometry [55 (link)].

For all assays, 1.5 × 10⁵ hTERT-BJ1 cells per well were seeded in 12-well plates. When cells were attached, drug treatments were added for 24, 48 or 72 h in triplicate. Reactive oxygen species (ROS) production was measured using CM-H₂DCFDA (C6827, Invitrogen). Cells were incubated for 20 min at 37°C with 1 μM CM-H₂DCFDA diluted in PBS, and then placed in complete media for 20 min at 37°C in the dark, to render the die fluorescent, according to the manufacturer. Autophagy Detection Kit (ab139484, Abcam) was used to detect autophagic vesicle production. Cells were trypsinized and incubated for 30 min at RT in the dark with a solution containing 1:1000 of Green Detection Reagent and 1:1000 of Hoechst in phenol-red free DMEM (D5921, Sigma) supplemented with 5% FBS, according to the manufacturer. Senescent cells were detected using the fluorescent senescence-associated β-galactosidase (SA-β-gal) activity marker C₁₂FDG (F1930, Molecular Probes). Cells were trypsinized and incubated with 2 mM of FDG substrate at 37°C for 1 minute. Staining was terminated by hypotonic shock by diluting the cells into ice-cold isotonic medium, according to the manufacturer.
ROS signal, autophagy vesicle signal, and SA-β-gal activity signal were quantified as mean fluorescent intensity of the viable cell population in a BD LSRII flow cytometer (BD Bioscience). Results were analyzed using FlowJo software.

Autophagosomes were detected by Autophagy Detection Kit (Abcam, Italy). The protocol adapted for immunofluorescence microscopy was applied for the staining of the autophagy vacuoles (green). The cells were grown on 4-wells chamber-slides (150,000 cells/well). Hoechst dye was used for nuclear marking (blue). Images were obtained by Nikon (Vico, Eclipse Ti80, Tokyo) under 60X magnification, using oil immersion objective. Percentage of necrotic cells was determined by calcein-AM/propidium iodide (PI; Sigma-Aldrich) staining using flow cytometry (Becton and Dickinson, Heidelberg, Germany). Cells were incubated for 30 min with calcein-AM (1 mg/ml) and PI (10 mg/ml). Minimum 20,000 events were acquired for each sample. The percentage of necrotic, PI positive, cells was distinguished from the total cell population.