Coolsnap es photometrics camera

The effects of ALA/light stress on migration properties of PC3 and DU145 cells was assessed by a gap closure (also known as “wound healing”) assay, which measures departure of cells from the general population on a flat surface [23 (link)]. Cells were seeded in 35-mm dishes and allowed to grow to at least 90% confluency, then sensitized with ALA-generated PpIX as specified in Sect. 2.3. After sensitization, a linear scratch was produced midway across the monolayer, using a sterile 200 μl pipette tip. The cells were then irradiated for 15 min (~1 J/cm² light fluence) in the absence vs. presence of an iNOS inhibitor and placed back in the incubator. At various times up to 48 h, cells in and around the gap region were observed and photographed using a Nikon Eclipse TS100 microscope with CoolSnap ES Photometrics camera and MetaVue software from Molecular Devices (Sunnyvale, CA). Extent of gap closure relative to a dark control and how iNOS inhibition affected this was determined by analysis of representative images at each post-irradiation time point [23 (link)]. For each reaction condition, data were obtained from at least six separate experiments.

A gap closure or “wound-healing” assay was used to analyze the migration rate of glioblastoma cells that survived ALA/light-induced stress and how this rate was affected by NOS inhibition. Cells were grown to ~90% confluency on 35-mm dishes. After ALA treatment, a linear scratch was made across the monolayer, using a 200 μl pipet tip. Cells were then irradiated, washed, and returned to the incubator. The gap zone was photographed before and after irradiation, using a Nikon Eclipse TS100 microscope equipped with a CoolSnap ES Photometrics camera and MetaVue software (Molecular Devices, Sunnyvale, CA). Extent (%) of gap closure in the absence vs. presence of a NOS inhibitor was determined using the following equation: 100 x [time-0 gap − time-t gap]/time-0 gap. A non-irradiated control was analyzed similarly. Data were obtained from at least four replicate experiments for each experimental condition.