Te2000e fluorescence microscope

Cell viability was assayed with the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen). VM-M3 cells were seeded for 24 hours on 18-mm glass coverslips in 22-mm 12-well plates at a density of 20,000 cells/well. The culture media was then replaced and treatment applied for 24 hours. Following the 24-hour treatment, cells were washed with D-PBS (Gibco, Life Technologies) and then incubated with 800uL of 2uM Calcein AM and 4uM Ethidium Homodimer-1 (EthD-1) in D-PBS for 30 minutes. Coverslips were then inverted and mounted onto glass microscope slides and cells visualized with a Nikon TE2000E fluorescence microscope and a 10X objective lens. Calcein-AM readily passes through the membrane of intact cells and is digested by cellular esterases that yield a fluorescent calcein product (Ex/Em: 495/515 nm) that can be detected with a FITC filter as an indicator of live cells. EthD-1 (Ex/Em: 525/590 nm) is cell-impermeable but emits a red fluorescence upon association with nucleic acid following loss of membrane integrity that can be detected with a TRITC filter as an indicator of dead cells. The live/dead ratios of 10 distinct fields of view were determined via direct cell count for each treatment.

Oxidative lipid damage was measured using the phenylbutadiene-based reporter for lipid peroxidation, BOPIDY® 581/591 (Molecular Probes) per the ascribed Image-iT® Lipid Peroxidation Kit protocol (Life Technologies). 50,000 VM-M3 cells were seeded on 18-mm glass coverslips in 22-mm 12-well plates for 24 hours. Culture media was then replaced and treatment applied. 10uM of the lipid peroxidation sensor was added to each well and cells incubated for 30 minutes. Cells were then washed three times with PBS and visualized with a Nikon TE2000E fluorescence microscope and a 10X objective lens. The ratio of oxidized (Ex/Em: 488/510nm, FITC filter) to reduced (Ex/Em: 580/590nm, TRITC filter) sensor fluorescence was determined for each cell in 5 distinct fields of view as an indication of the extent of lipid peroxidation induced by each treatment.

Cell migration and invasion assays were performed using transwell chambers with 8 μm pore-size polycarbonate filters, noncoated (migration) or precoated with BME (invasion) according to manufacturer’s instructions. Cells were starved in serum-free media for 24 h and detached. For the invasion assay, the upper compartment was pre-coated with BME to form a matrix barrier in a 24-well plate. RPMI medium containing 10% FBS (0.5 mL) was added in the lower chamber to induce migration and invasion. Following 24 h (migration) or 48 h (invasion), the migrating/invading cells present on the lower surface of the filter member were stained with Calcein AM and incubated for 2 h at 37 °C in a cell culture incubator. Migrating/invading cell-associated fluorescence intensities were measured with a FluorStar Omega microplate reader using 485 nm excitation and 520 nm emission. The number of migrating/invading cells on the lower surface of the filter membrane was also observed and photographed using a Nikon TE-2000-E fluorescence microscope. The percent migration/invasion of miR-214KO cells was calculated using the formula (miR-214KO/WT) × 100. All analyses were performed in duplicate with three independent experiments.