Immunofluorescence Analysis of HCT116 Cells

HCT116 (5 × 10⁴ cells/well) were seeded on sterilized coverslips contained in six-well plates, which were incubated at 37 °C with 5% CO₂ for 24 h. The cancer cells were treated with various conditions, including a control (non-treated cells), conditioned media, and 20 μM H₂O₂ as a positive control, and incubated at 37 °C with 5% CO₂ for 48 h. The treated cells were fixed with 4% paraformaldehyde and washed with PBS. The cells were permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, USA), and then primary antibody (mouse anti-human E-cadherin (1:1000) and rabbit anti-human TrxR-1 (1:1000) (Abcam, USA)) were added. The cells were incubated at 4 °C overnight, and they were then washed with PBS in triplicate. The secondary antibody (goat anti-mouse Alexa488, goat anti-rabbit Alexa488 (1:1000) (Abcam, USA)) was added and incubated for 90 min at room temperature. The protein expression was captured under a fluorescence microscope [22 (link)].