Fv1000 asw

As described by Katsori et al.^{44 (link)}, NF-κβp65 signaling pathway was determined in murine pancreatic cancer cell line (Pan02) under the effect of 1-MT or without 1-MT. briefly, Pan02 cells were cultured in RPMI 1640 (Key GEN Bio, China) supplemented by 10% Fetal Bovine Serum (Hycone, Australia), and 1% penicillin and streptomycin (Gibco, USA) for 24 hours. Then 1 × 10⁴ cells co-cultured with/without 10 ng/ml of 1-MT. 24 hours later cells were washed with phosphate-buffered saline (PBS) then cells fixed and permeabilized by 4% formaldehyde in PBS. Next cells were blocked in 5% milk–10%, Triton X-100 in PBS for 1 h at room temperature. Then p65 visualized by purified Anti-NF-κβp65 (ab32536) at 1:100 dilutions for 12 hours. After that cells were washed and probed by 1:500 dilution of Alexa Fluor 488 goat anti rabbit IgG for 2 hours. Later, DAPI nuclear counterstain was performed for 10 min. Finally, cells were washed by PBS and scanned by laser confocal microscopy FV1000-ASW (Olympus, Japan).