Evos digital color fluorescence microscope

Untreated and doxycycline-treated DIE cells were stained with AnnexinV-Alexa Fluor 488 and PI (Thermo Scientific), by adding the staining solutions directly to growth medium at a 1:50 and 1:100 ratio respectively. Cells were incubated overnight in growth medium containing staining solution(s) and with or without doxycycline treatment. Live imaging during treatment of these cells was carried out using a Confocal microscopy (Zeiss LSM 700). Still images were taken after the overnight incubation with an EVOS Digital Color Fluorescence Microscope (Invitrogen).

Transwell migration assays were performed using Transwell plates with 0.8 µm pore polycarbonate membranes (Corning Transwell, Sigma-Aldrich). Three days post-siRNA transfection, MDA-MB-231 cells were seeded in the upper chamber without FBS and allowed to invade to the reverse side of the chamber under chemoattractant conditions with 10% FBS-containing medium in the lower chamber. Following incubation for 16 h at 37°C, the cells were fixed with 3.7% formaldehyde for 2 min at RT. Cell permeabilization was carried out by incubation in 100% methanol for 20 min at RT. Cells were then stained with Giemsa for 15 min at RT. The final total cell number between conditions was always checked by wide-field microscopy to avoid proliferation bias for migratory cell comparison. Non-migrated cells were finally removed from the upper chamber using a cotton swab. Migrated cells adhering to the underside of the chamber were photographed using a light microscope at 200× magnification (Invitrogen EVOS Digital Color Fluorescence Microscope). Cell counting was performed with ImageJ in ten different fields per condition (Schneider et al., 2012 (link)). Three independent experiments were performed for each condition.