Confocal software lcs version 2.61 build 1537

The cells were grown in the chamber slides. Confocal laser scanning microscopy was performed at the CNSI Advanced Light Microscopy/Spectroscopy Shared Resources Facilities at UCLA with technical assistance from Dr. Matthew Schibler. Briefly, images were captured using the Leica TCS-SP2-AOBS confocal microscope with x63 oil objective under different gain settings. The 488-line argon laser was used to capture GFP staining, and the diode 405 nm laser was used to capture DAPI nuclear stain. For mCherry staining, 594-line HeNe laser was used. Image acquisition was performed using Leica Confocal Software (LCS) version 2.61 Build 1537. Scans were performed with pinhole set at Airy unit 1.5. SmartGain were adjusted for each sample for optimal brightness. Fluorescent images of cells were taken as single channel images then converted to overlay images and all images were saved in TIFF format.

Images were captured using the Leica TCS-SP2-AOBS confocal microscope with x63 oil objective, using identical magnification and gain settings, as previously described (8 (link), 29 ). The 488-line argon laser was used at 667 V to capture the BODIPY® FL C₁₆ lipid stain; the diode 405 nm laser was used at 436 V to capture DAPI nuclear stain. Image acquisition was performed using Leica Confocal Software (LCS) version 2.61 Build 1537. Fluorescent images of cumulus cell lipid content were quantified by one of two observers using ImageJ (http://rsbweb.nig.gov/ij/) to determine mean fluorescence (fluorescence/unit area) of 20 cells per patient. The reproducibility of quantitative cellular lipid measurements made by the two different observers measuring the same lipid quantity was large (0.96 [95% CI 0.86–0.99]), indicating a very high degree of consistency between the two observers.
Images taken as single channel images were converted to overlay images and all images were saved in Tag Image File Format (TIFF). Single channel images of BODIPY® FL C₁₆ and DAPI were used to create an overlay image, with single channel BODIPY® FL C₁₆ used for lipid quantification in ImageJ. Background staining was accounted for by measuring five negatively stained regions per cell, which were subtracted from the total mean fluorescence.