Edu alexa fluor 555 imaging kit

The Real-Time polymerase chain reaction (qPCR) system was purchased from Applied Biosystems (Foster City, CA, USA). Click-iT Nascent RNA Capture kit was purchased from Invitrogen (Carlsbad, CA, USA). Western blot analysis-related equipment was purchased from Invitrogen. EdU Alexa Fluor 555 Imaging kit was purchased from Invitrogen. Tetramethylrhodamine methyl ester (TMRE) was purchased from Molecular Probes (Eugene, OR, USA). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), cycloheximide (CHX), and actinomycin D were purchased from Sigma (St. Louis, MO, USA). BSA and AGEs-BSA were obtained from Merck-Millipore (Darmstadt, Germany). A fluorescence microscope (CKX41-F32FL) was purchased from Olympus (Tokyo, Japan). Microchemi 4.2 was purchased from DNR Bio-Imaging Systems, Ltd. (Jerusalem, Israel). Transwell-related equipment (8-µm pore) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Microplate reader was purchased from Bio-Rad (Hercules, CA, USA). Antibodies for BAG3 (sc-292154) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-47724) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lentiviral vectors were purchased from GeneChem (Shanghai, China).

Min6 cell proliferation was detected by two EdU methods (with TMB coloration and fluorescent dye), and the primary islet proliferation was detected by EdU with fluorescent dye. Briefly, Min6 cells were incubated with ELVs for overnight, and10μM EdU was added 2 h before cells were fixed. Islets were seeded onto Matrigel (#356230, Corning, NY, US) coated coverslips to help the adherence and growth of islets, and incubated with ELVs and 10 μM EdU for 48 h. EdU was detected using EdU Cell Proliferation Kit with TMB (Beyotime Institute of Biotechnology) or EdU AlexaFluor555 Imaging Kit (Invitrogen, CA, US). In the latter assay, cells and islets were counterstained with anti-insulin antibody (#ab181547, Abcam, Shanghai, China) and fluorescent secondary antibody (#4412, Cell Signaling Technology, Shanghai, China) to define β-cells and islets.