Ix71 inverted microscope

Apoptosis and autophagy were evaluated with Olympus IX71 inverted microscope using Multi-Parameter Apoptosis Assay Kit (Cayman chemical, Ann Arbor, MI, USA, cat no 600330), respectively, Autophagy/Cytotoxicity Dual Staining Kit (Cayman chemical, Ann Arbor, MI, USA, cat no. 600140) according to the manufacturer’s protocol. Cells were cultured at a seeding density of 1 × 10⁴ cells in 8-well chamber slides and transfected with 50 nM of miR-29b-3p inhibitor and negative control inhibitor for 48 h. To evaluate the apoptotic effect, cells were stained with Annexin-V FITC used to identify different types of cell death (early and late apoptosis) and evaluated at 485/535 nm. PI (propidium iodide), which is a DNA-binding dye molecule, is used to differentiate late apoptosis from necrosis being evaluated at 535/617 nm. The autophagic vacuoles were stained with monodansylcadaverine (MDC) and visualized in UV. Cellular death was evaluated at 520/610 nm using PI (propidium iodide).

Autophagy effects were evaluated with Olympus IX71 inverted microscope (Shinjuku-ku, Tokyo, Japan) using Autophagy/Cytotoxicity Dual Staining Kit (Cayman cat no. 600140) according to the manufacturer’s protocol. Thus, cells were plated at a density of 8 × 10⁴ in a 96-well plate and treated with the abovementioned concentrations. Forty-eight hours after therapy, autophagic vacuoles were stained with monodansylcadaverine (MDC) and observed under UV light. Cell death was assessed using PI at 520/610 nm.

Mitochondrial activity was evaluated with Olympus IX71 inverted microscope (20X magnification) using Multi-Parameter Apoptosis Assay Kit (Cayman chemical, Ann Arbor, MI, USA; catalog no. 601280). At a seeding density of 1 × 10⁴, cells were seeded in 8-well chamber slide and transfected with 50 nM of miR-29b-3p inhibitor and negative control inhibitor for 48 h. Post-transfection, cells were stained with TMRE (Tetramethylrhodamine ethyl ester) and visualized at 560/595 nm to determine mitochondrial membrane activity potential. Additionally, cells were also stained with Hoechst and visualized in UV.