Enzo cyto id autophagy detection kit

To detect apoptotic cells, a sample of 1 × 10⁵ MM cells/mL (RPMI8226, CAG, or MM.1S) was plated in 12-well plates and incubated with Dex with or without CQ or AZD6738 for 48 h. The cells were harvested, washed twice with phosphate-buffered saline (PBS), resuspended in 100 μL of staining buffer and stained with Annexin V–APC/PI according to the manufacturer’s instructions. The cells were detected using flow cytometry, and the data were analyzed using FlowJo 7.6.1.
RPMI8226 and CAG cells (2 × 10⁵ cells/well) were cultured with Dex (0, 50, or 100 μM) in six-well plates for 48 h, washed twice with PBS and permeabilized with precooled 75% ethanol at 4 °C overnight. The next day, the cells were washed twice with PBS and incubated with 500 μL of PI for 30 min at room temperature according to the manufacturer’s instructions. The cells were detected using flow cytometry (BD Biosciences, CA, USA), and the data were analyzed using ModFit software (version 3.2, Verity Software House).
RPMI8226 cells (1 × 10⁵ cells/ml) were treated with Dex (100 μM) in 12-well plates for 48 h. The cells were harvested, and autophagy was detected by flow cytometry with Enzo Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, NY, USA) according to the manufacturer’s instructions.

The Enzo CYTO-ID autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA) was employed following the manufacturer’s instructions. Briefly, 2 × 10⁴ INS-1E cells were seeded on a 96-well plate (Nunc^TM, Roskilde, Denmark). After DMT1 siRNA transfection, the cells were treated with or without 50 pg/mL of recombinant rat IL-1β for 24 h. After washing with the buffer provided with the kit, the kit fluorescence buffer was added for 30 min at 37 °C in the dark. The fluorescence intensity (FITC: emiision~530 nM excitation~480 nM and Hoechst: emiision~480 nM excitation~340 nM) was detected using FLUOstar^® Omega plate reader (BMG Labtech, Aylesbury, UK).

Apoptosis and cell cycle assays were conducted as previously described [16 ]. ARP-1 cells (1 × 10⁵ cells/ml) were treated with HCQ (10 μM) in 24-well plates for 24 h. The cells were harvested, and autophagy was detected by flow cytometry with an Enzo Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, NY, USA) according to the manufacturer’s instructions.