Ix51 inverse fluorescence microscope

Keratinocytes, hrHPV + KCs, control shRNA-expressing KCs, or IFITM1 shRNA-expressing KCs were seeded 5000 cell/well in 96-well plates and allowed to attach overnight. Cells were cultured in presence of indicated concentrations of IFNγ and/or TNFα in 150 μl for 96 h. 15 μl/well MTT (3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyl-2H-tetrazolum bromide) stock solution (5 mg/ml in 0.1M PBS) was added for 3 h. When the purple formazan precipitate was clearly visible under the microscope, bright light pictures were made using an Olympus IX51 inverse fluorescence microscope (Olympus, Zoeterwoude, The Netherlands). Images were captured by ColorView II Peltier-cooled charge-coupled device camera (Olympus) and archived using Cell^{^}F software (Olympus).

Two days post-transduction, HeLa cells were trypsinized, resuspended in fresh culture medium and subsequently placed back in the incubator for 24 h before imaging. The DNA dye Hoechst 33342 (Thermo Scientific Cat. Nr: 62249) was diluted 1:10.000 in the culture medium, and the cells were then incubated at 37 °C in 10% CO₂ for 10 min. Total and transgene-positive cells present in randomly selected microscopy fields were detected through fluorescence microscopy directed to the DNA dye and EGFP, respectively, by using an Olympus IX51 inverse fluorescence microscope equipped with an XC30 Peltier CCD camera and the CellF software (both from Olympus). Myoblasts were transduced as described above, and at 3 days post-transduction, the myoblasts were tripsinized and transferred to wells of 24-well plates. Bright-field images were acquired 2 days after re-seeding the cells by using an inverted Leica DMi8 microscope equipped with a DFC 450c camera, 40 × objective and the LAS 4.8.0 software (both from Leica).