Eclipse ts100 inverted fluorescence microscope system

The aptamer-mediated cell binding was carried out as previously reported [20 (link)]. One hour after incubation with indicated aptamers at 200 nM concentration in wells of a 24-well plate, 1 µL of Hoechst 33342 solution (1 µg/mL) was added to the plated cells and incubated for 10min at 37 °C in a humidified incubator supplying 5% CO₂/air. The cells were then washed prior to visualization using a Nikon Eclipse TS100 Inverted Fluorescence Microscope system (Nikon).

Apoptosis was assessed using ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Darmstadt, Germany, S-7165) in accordance with the manufacturers’ instructions. Briefly, cells were seeded in an 8-chamber slide (Lab-Tek, Campbell, PM, USA, 177402) at 20,000 cells/chamber 24 h before the test. After incubation, cells were treated with 500 nM of either CL-4 or CL-4RNV616 aptamer in DMEM medium for 72 h. After PBS washing, the cells were then fixed in 1% paraformaldehyde in PBS for 10 min at room temperature followed thorough wash with PBS. Then, the slide was incubated with 55 μL/5 cm² of working strength TdT enzyme at 37 °C for 1 h. One hour later, the reaction was stopped and washed with stop/wash buffer at room temperature for 10 min. After thorough washing in PBS, the sections were then incubated with rhodamine-conjugated anti-digoxigenin antibody (1:50) at room temperature in a darkened humidified chamber for 30 min. After another round of thorough washing with PBS, the sections were counterstained with Anti-fade mounting medium (ThermoFisher, Waltham, USA, P36930) containing 3 μg/mL Hoechst 33342 (Sigma, Darmstadt, German, B2261) before visualization under a Nikon Eclipse TS100 Inverted Fluorescence Microscope system (Nikon, Tokyo, Japan).