Dcf 420 c camera

HEK293T cells were transfected with GFP or GFP-WT1 as described above. Cells were grown to confluence in 6-well dishes and a wound was made using a pipette tip. Cells were washed with PBS and fresh media was added to each plate. Dishes were visualized at 40× magnification and photographed at t = 0 and 24 hr after the wound was made using a Leica DM IL microscope equipped with a DCF 420 C camera and Leica Application Suite Software (Leica Microsystems GmbH, Wetzlar, Germany). Images were processed using Photoshop (Adobe Systems Inc., San Jose, CA). Wound coverage was reported by measuring the distance between the scratch borders at t = 0 hr and t = 24 hr.

Senescence-associated β-galactosidase (SA-β-gal) activity was detected as previously described [34 (link)]. Briefly, 1 × 10⁴ cells/cm² were seeded in triplicate into 12- to 24-well plates. For knockdown experiments, the cells were transfected immediately with siRNA. After overnight attachment, cells were treated daily with either DMSO or 0.25–5.0 μM RSL. Six hours after the last dose, cells were washed with PBS, fixed for 15 min at RT with a 2% formaldehyde/0.2% glutaraldehyde solution, then incubated overnight at 37 °C in a non-CO₂ atmosphere with X-Gal staining solution (40 mM citric acid pH 6.0, 5 mM potassium hexacyanoferrate II, 5 mM potassium hexacyanoferrate III, 150 mM NaCl, and 2 mM MgCl₂) containing 1 mg/ml X-β-gal substrate (Carl Roth, Karlsruhe, Germany). Cells were extensively washed with PBS and imaged with a Leica DMI6000B inverted microscope equipped with a DCF420 C camera and LAS X Core software (Leica Microsystems, Wetzlar, Germany). The percentage of SA-β-gal-positive cells from total cells was calculated for three biological replicates. Images were captured using a Zeiss Axio Imager Z1 microscope equipped with an AxioCam HRc camera and AxioVision 4.8 software (Carl Zeiss, Oberkochen, Germany).