96 well flat bottom tissue culture plate

For OD600 kinetics assays with taurocholate in 96-well plates, ~1.6 x 10⁷ spores (0.55 OD600 units) for each condition tested were resuspended in BHIS and 180 μL were aliquoted into two wells of a 96 well flat bottom tissue culture plate (Falcon) for each condition tested. The spores were exposed to 1% taurocholate, or water (untreated) in a final volume of 200 μL. The OD600 of the samples was measured every 3 minutes in a Synergy H1 microplate reader (Biotek) at 37°C with constant shaking between readings. The change in OD600 over time was calculated as the ratio of the OD600 at each time point to the OD600 at time zero. These assays were performed on three independent spore preparations. Data shown are averages from three replicates.
For phase contrast microscopy, purified spores were resuspended to an OD₆₀₀ of 0.5 in BHIS ± 1% taurocholate and incubated aerobically for 90 min at 37°C. Samples were spun down (5,000 x g, 10 min, 4°C) washes with PBS and fixed with 3.7% paraformaldehyde for 15 min at RT. Following fixation, samples were washed with PBS and mounted on agarose pads.

UMSCC-10B and UMSCC-22B cells were plated into a 96-well flat-bottom tissue culture plate (Falcon) at a density of 5,000 cells per well. After a 24 h plating period, these cells were transfected with the miRNA expression plasmids or inhibitors. For cisplatin sensitivity experiments, the control and transfected cells were subsequently exposed to one of several doses of cisplatin ranging from 0–5 μg/mL. After a 48 h incubation period, cellular proliferation was analyzed using an MTS proliferation assay (Promega) in accordance with the manufacturer's protocol. All assays were performed in triplicate wells and experiments were individually performed at least twice.