Evos m5000 imaging fluorescence microscope

The analysis was conducted following the previous study [26 (link)]. In brief, 1 × 10⁴ cells/well were seeded in eight well chamber slides and incubated for 24 h in a humidified environment before SCF sample treatment. Wells were rinsed with PBS and fixed with 4% formaldehyde after 30 min of TNF-α/IFN-γ stimulation. After PBS washing, cells were incubated for 1 h in blocking buffer (PBS containing 5% normal goat serum and 0.3% Triton^TM X-100). The wells were treated for 2 h with Alexa Fluor® 488 conjugated Anti-Mouse IgG after being incubated overnight with a primary antibody (anti-NF-kB p65). Followed by PBS washing, slides were covered with coverslips using Prolong^® Gold antifade reagent with DAPI, and cells were visualized using a Thermo Fisher Scientific EVOS M5000 Imaging fluorescence microscope.

The RAW 264.7 macrophages (1 × 10⁴ cells/chamber) were cultured in chamber slides and incubated for 24 h before sample treatment. After 30 min of LPS stimulation, wells were washed with PBS and fixed with 4% formaldehyde. Before being incubated with primary antibodies (anti-NF-κB p65) overnight, cells were blocked using a blocking buffer (PBS containing 5% normal goat serum and 0.3% Triton^TM X-100) for 1 h. The cells were then treated for 2 h with Alexa Fluor^® 488 conjugated anti-mouse IgG. The slides were covered with coverslips using Prolong^® Gold antifade reagent with DAPI after PBS washing, and cells were visualized using an EVOS M5000 Imaging fluorescence microscope from Thermo Fisher Scientific.