Slidebook software

Cells were seeded in wells of a Lab-Tek II CC2 glass chamber and grown to reach a 70% level of confluence. Cells were fixed for 10 min in 4% para-formaldehyde at 25°C, permeabilized for 5 min in 0.25% Triton X-100, and blocked for 1 h in 2% BSA at 25°C. Cells were incubated for 1 h with the respective primary antibodies followed by incubation (30 min; 25°C) with species-specific secondary antibody. Cells were mounted on slides using the Prolong Gold reagent (Life Technologies) containing 4',6-diamidino-2-phenylindole (DAPI). Confocal microscopy was conducted using a LSM 710 NLO Zeiss Multiphoton Laser Point scanning confocal microscope equipped with a multi-photon Mai-Tai laser HB–DeepSee system (690–1024 nm). Images were acquired using ZEN software (Zeiss, Thornwood, NY). Alternatively, a Spinning Disc confocal microscope DSU-IX81 (Olympus, Waltham, MA) was used and images were captured using Slidebook software (Olympus). Images were further processed using ImageJ (http://www.macbiophotonics.ca). Our experiments were performed multiple times and at least three images were obtained from three independent biological replicates. Colocalization analysis was conducted as previously described [24 (link)]. The data were analyzed using Statview software (SAS Institute, Cary, NC; http://www.statview.com). The p-values below 0.05 were considered significant.

BAECs were injected into Sykes-Moore chambers with a 1 mm thick gasket and maintained in DMEM supplemented with 10% FBS and 10 mM HEPES. Time-lapse recording was carried out every 2 min using an Olympus IX81 inverted microscope with a CO₂-humidity-temperature controlled incubation chamber (Precision Plastics, Beltsville, MD, USA), a 20× phase contrast objective lens (N.A. 0.40), and a deep-cooled CCD camera (Orca-ER, Hamamatsu Photonics, Hamamatsu, Japan). Cells were first recorded for 1 h in the right-side up orientation, flipped upside down by inverting the chamber, quickly identified, and further recorded for 1 h. Cell tracks were made by connecting the centroid position of cells and analyzed using Olympus SlideBook software. The experiments were repeated three times.

Non-transduced RH30 or RH4 cells were seeded onto a 24-well plate at 200 000 cells per well. Growth media was replaced with media containing increasing concentrations of LiCl (10 mM, 20 mM or 40 mM) or AR-A014418 (2 μM, 10 μM or 20 μM) and incubated for 24 h at 37 °C and 5% CO₂. Alternatively, stably transduced RH30 or RH4 cells were seeded onto a 24-well plate at 300 000 cells per well. A 100 μl gel-loading tip was used to generate a scratch in the monolayer, the cells were carefully washed twice with 1 × phosphate-buffered saline to remove floating cells, and subsequently incubated with fresh media containing the GSK3β inhibitors or non-supplemented media, respectively. Images were captured at exact spots every 15 min for 6 h using an Olympus time-lapse microscope (Olympus, Pittsburgh, PA, USA) with subsequent analysis using SlideBook software. Sixty to 80 individual cells from four independent images were tracked and the velocity was determined for each cell using ImageJ software.

After L3 stage worms were infected with C. albicans WT-OXYellow [17 (link)] and allowed to incubate overnight. They were washed with 1 ml of sterile M9W and collected by centrifugation at 1000 rpm. The wash was repeated three times. The worms were then paralyzed with 1mM tetramisole for 30 minutes. Anesthetized worms were mounted on 2% agarose pads and imaged using an Olympus IX81 automated inverted microscope and Slidebook software (Version 6.0). Imaging and photography was performed using FITC and TRITC filter sets to capture CTA1-yEGFP and ADH1-yCherry expression, respectively. The FITC and TRITC images were used to quantify expression of CTA1-yEGFP in comparison to ADH1-yCherry to obtain a normalized measurement of ROS production. The amount of fluorescence for each filter set was quantified using ImageJ 1.48 freeware. The fluorescence was measured in the worm intestine within a defined area. The same area of background was subtracted. The ratio of the FITC to the TRITC measurements was calculated. A total of 28–30 worms were measured and averaged for each strain and the standard error was calculated. Statistical differences were determined by an unpaired t-test using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA). Each experimental condition was compared pairwise to the control condition. P-values < 0.05 were considered to be statistically significant.