Slide flasks

The human follicular thyroid carcinoma cell line FTC-133 was cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin/streptomycin (Life Technologies) at 37 °C and 5% CO₂ until use for the experiment. For RPM experiments FTC-133 cells were seeded at a density of 1 × 10⁶ cells per flask either in T25 cell culture flasks (Sarstedt, Nümbrecht, Germany) for mRNA and protein extraction or in slide flasks (Sarstedt) for immunofluorescence staining. Cells were given at least 24 h to attach to the bottom of the flasks.

The Click-iT™ Plus TUNEL assay (Invitrogen) was used for apoptosis detection. FTC-133 cells were cultured in slide flasks (Sarstedt) under static culture conditions or exposed to the RPM, supplemented without or with 1000 nM DEX. After 4 h or 3 days cells were fixed with 4% PFA and prepared for the evaluation of apoptosis. The staining procedure was performed according to the manufacturer’s recommendation. A positive control sample was treated with DNase I (Epicentre, Madison, WI, USA) to induce DNA fragmentation. The stained cells were examined using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss) equipped with an external light source and an objective with a calibrated 630× magnification.