Axiocam 506 ccd camera

Irradiated IN528 cells were subjected to alkaline and neutral comet assays (single-cell gel electrophoresis) for detecting DNA single- and double- strand breaks, respectively, as previously described32 . In brief, irradiated cells were washed with PBS and mixed with low melting agarose (1:10), before being loaded onto microscope slides. Cell lysis was performed at 4 °C (alkaline comet assay) or 37 °C (neutral comet assays). After electrophoresis for 25 minutes at room temperature, DNA was stained with propidium iodide and imaged with an AxioImager A1 fluorescence microscope (Zeiss) equipped with an AxioCam 506 CCD camera (Zeiss). The cell number with DNA comets and the DNA percent content in comet tail region were measured using ImageJ and OpenComet 1.3 software (three assays, each with about 200 cells analyzed).

A mixture of 1.5 μl of Lipofectamine 2000 (Invitrogen) and 2.0 μg of pAAV.CAG.SF-iGluSnFR.A184S (Addgene plasmid #106198; gifted from Loren Looger; http://n2t.net/addgene:106198; RRID: Addgene_106198) in 60 μl Neurobasal Medium (Invitrogen) was prepared and incubated at room temperature for 30 min. Media from neuron cultures was saved aside and cells were incubated with this mixture at 37°C and 5% CO₂ for 30 min. Mixture was aspirated and replaced with original media. Time lapse wide field fluorescence videos were obtained at five frames per second for 60 s using a Zeiss Laser Scanning Microscope (LSM) 800/Airyscan equipped with a Colibri 7 LED light source, Zeiss Axiocam 506 CCD camera, and 63×/1.4 NA objective lens at 37°C and 5% CO₂. Neurons were imaged in their culture media.

HUVECs of the third passage were cultured in 0.2% gelatin-coated 24-well plates in EGM-2MV growth medium supplemented with 2 mM glutamine, 40 µM carnitine, 100 U/mL penicillin, and 100 μg/mL streptomycin. PA was added to achieve 0.5, 0.75, 1.0, or 1.5-mM final concentration. Where applicable, AICAR was always present at 1 mM final concentration. The media were exchanged daily. Phase-contrast microscopy was performed using an Axio Observer A1 (Zeiss, Germany) inverted microscope equipped with an on-stage thermostat (37 °C) and an AxioCam 506 CCD camera (Zeiss, Oberkochen, Germany). Images were acquired and processed using the AxioVision 4.8.2 software (Zeiss, Germany) and the ImageJ freeware (NIH, Bethesda, MD, USA).