Ti e wildfield inverted microscope

MX1 was knocked down in two colorectal cell lines, DLD1 and SW450, using commercially available siRNA reagent (sc45260, Santa Cruz Biotechnology, CA) with DharmaFECT transfection reagent (Thermo Fisher) following manufacturer's instructions. The wound-healing assays were performed as previously described (18). For each transfection reaction, including controls mock-transfected with reagent only, multiple replicate wells of a 96-well tissue culture plate were seeded with approximately 5000 cells and incubated for 3 hours to allow the cells to attach. Wounds were created after 24h by manually scratching each well with a yellow pipette tip and the plate was then gently washed with pre-warmed medium to remove detached cells and imaged on a Nikon Ti – E wild field inverted microscope using scan large image option at 10x magnification. The plate was then incubated for 24 hours and imaged again. The images were processed by the NIS Elements software, to calculate wound closure rate (LUNDEBERG et al.,1992) and determine statistical significance whenever judged necessary. After being imaged the cells were lysed, separated by SDS-PAGE and transferred to PDVF membrane. Anti- MX1/2/3 (C1) mouse monoclonal antibody (Santa Cruz Biotechnology, sc-166412) was used for detection of MX1. Imaging was done using Li-Cor infrared Odyssey system.