Spectramax gemini em microplate spectrofluorometer

HT-29 and Caco2 cells were seeded with a cell number of 3 × 10⁵ per well in 24-well culture plates. After 24 h, HT-29 and Caco2 cells were cultured and treated as described (2.2. Cell lines culture). Cells were washed with PBS three times and fixed in 4% paraformaldehyde (methanol-free, Thermo Fisher) for 10 min, permeabilized with 0.1% Triton X-100, washed in PBS, and blocked for 30 min in 1% BSA and 5% goat serum for 1 h at 25 °C. Then, they were rinsed with PBS three times for 10 min each. After the cells were blocked, they were incubated for 30 min at 25 °C with Alexa Fluor 488-conjugated phalloidin (Invitrogen, CA, USA) to visualize F-actin. The samples were examined by fluorescence microscopy (100×, Axio Observer.D1, Carl Zeiss), and the amount of F-actin was determined using a SpectraMax GEMINI EM microplate spectrofluorometer (Molecular Devices) at 485 nm (excitation wavelengths) and 525 nm (emission wavelengths). The fluorescence intensity was calculated by the relative fluorescence of each treatment compared to control cells. The experiments were performed in triplicate.

MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen, Grand Island, NY) was used to quantify mitochondrial superoxide and performed according to manufacturer instructions. Briefly, after indicated treatment, cells were stained with MitoSoX Red (5 µM) for 10 min in HBSS media at 37 °C and fluorescence was measured using a SpectraMax Gemini™ EM Microplate Spectrofluorometer (Molecular Devices, San Jose, CA) as described^{15 (link)}.