Cyto id autophagy kit

Autophagy was measured using a CYTO-ID autophagy kit (Enzo Life Sciences, Exeter, UK). The CytoID detection reagent was diluted in the assay buffer, and NRVMs were loaded for 30 min at 37 °C before being washed and imaged on a fluorescence microscope. The CytoID reagent was excited at 488 nm, and emission was measured above 515 nm. Image collection and analysis were performed as described above.

OnTarget plus siRNA smart pools were from Dharmacon/Horizon Discovery, Waterbeach, UK. Individual mice cytokines were purchased from Immunotools, Friesoythe, Germany, and LPS was purchased from Invitrogen/Thermo Fischer Scientific, Waltham, MA, USA. Propidium iodide was from Calbiochem/Merck, Darmstadt, Germany, and Hoechst33342 from Thermo Fischer Scientific, Waltham, MA, USA. Rapamycin was bought from Hycult Biotech, Uden, Netherlands, AKT inhibitor VIII from MedChem Express, Monmouth Junction, NJ, USA and cycloheximide from Merck, Darmstad, Germany. L-Azido-homo-alanine (L-AHA) and Protein synthesis detection Kit were from JenaBioscience, Jena, Germany. Real-time Glo apoptosis/necrosis kit was purchased from Promega, Fitchburg, USA. Insulin, holo-transferrin, sodium selenite and dexamethasone were from Sigma-Aldrich/Merck, Darmstadt, Germany. Lipofectamine RNAimax reagent was purchased from Invitrogen. BCA assay kit was from Serva Electrophoresis GmbH. ECL-substrate solution was from Thermo Fischer Scientific. DAPI-containing fluoroshield mounting medium was from Sigma-Aldrich/Merck, Darmstadt, Germany. Cyto ID autophagy kit was purchased from Enzo life sciences, Lörrach, Germany.