Inverted epifluorescent microscope

KSY508 cells were grown overnight and placed on agarose pads made by melting 0.2 g of agarose in 1 ml -TRP media (Waddle et al., 1996 (link)). The cells were then viewed using the Olympus Inverted Epifluorescent Microscope with a 100× Plan Apo/NA 1.4 DIC Objective. An FITC filter (EX 482/35 506DM EM 536/40) was used (Brightline). Images were captured with a Hamamatsu ORCA285 CCD camera. Shutters, filters, and camera were controlled using SlideBook software.

Confocal Microscopy. For mitochondrial ROS production measurements using MitoSOX, 4×10⁴ cells were plated in 0.5 mL of media in 4-well Lab-Tek II Chambered #1.5 Coverglass and incubated for 24 h. To monitor cytoplasmic ROS production, cells were transiently transfected with pHyPer-cyto, a reporter for H₂O₂ levels in the cytoplasm, using FuGene6 Transfection Reagent according to manufacturer's protocol, and incubated for 24 h [58] (link). Where indicated, cells were pretreated with LPA or receptor antagonist before labeling with 1 µM MitoSOX for 10 min [59] (link). MitoSOX reagent was removed and cells were washed three times with media. The field of view was located and focused before the addition of the Taxol or cisplatin. Images were collected over time using Zeiss LSM510 laser scanning confocal microscope.
Fluorescent Microscopy. For measurement of long term ROS production, SKOV3 cells (5 × 10⁴) were plated in 2 mL RPMI supplemented with 10% FBS in 35 mm dishes. Cells were treated 24 h after plating with indicated concentrations of cisplatin for 24 h then incubated with dichlorofluorescein diacetate for 10 min, washed and visualized using an Olympus inverted epi-fluorescent microscope with FITC filters.