Millicell ez glass slide

Detection of apoptotic cells was performed using APO-BrdU^TM TUNEL assay kit (Invitrogen). Briefly, 3 × 10⁴ cells were plated in each well of an 8-well Millicell EZ glass slide (Millipore, Billerica, MA). After treatment with CDDP (10 μM) for 48 h, the cells were fixed in 4% (w/v) paraformaldehyde on ice for 15 min and then washed twice with PBS. The fixed cells were stored in 70% ethanol at 4°C overnight followed by nicked ends labeling reaction according to the manufacturer's instructions. The positively labeled cells (green fluorescence) were visualized by fluorescence microscopy.

A549 cells grown on Millicell EZ glass slide (Millipore) at 80% confluence were infected with WSN at an MOI of 2 for 4 to 8 h.p.i. or were transfected with 2 μg of the WSN NS1 clone. The cells were fixed in phosphate-buffered saline (PBS) containing 4% formaldehyde, permeated with 0.3% Triton X-100, blocked with 5% normal donkey serum for 1 h at 25°C, and then stained with anti-XRN1 (diluted 1:20; Santa Cruz), anti-WSN NS1 (diluted 1:100; NUS, anti-DCP1A (diluted 1:20; Santa Cruz), or anti-FLAG (diluted 1:100; Sigma) antibodies for 2 h at 37°C. Subsequently, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (diluted 1:200; green, Invitrogen), goat anti-rabbit IgG (diluted 1:200; red, Invitrogen), or donkey anti-goat IgG (diluted 1:200; purple, Invitrogen) for 1 h at 25°C. The cells were washed three times with PBS and mounted in Vectashield antifade mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Confocal images were obtained with a confocal laser-scanning microscope (Zeiss; LSM 700).