Axiovision fluorescence microscopy

Images of co-culture experiment were acquired with Zeiss Axiovision fluorescence microscopy. 8–12 images were acquired for each well at × 5 magnification. Images were analysed by Image J angiogenesis plugin (Gilles Carpentier, Faculté des Sciences et Technologie, Université Paris Est Creteil Val-de-Marne, France). Briefly, channels were split automatically. On the GFP channel, total tube length, branches number and total length of branches were acquired by Analysis HUVEC Fluo program. Each condition was carried out in triplicate.

Whole slides were scanned to acquired total images at × 10 magnification with a Zeiss Axiovision fluorescence microscopy. CD31 expression areas were analysis by Image J. Briefly, The image was converted into RGB stack format. CD31 staining was quantified by choosing threshold program and adjusting the threshold parameters. Once the threshold parameter was adjusted, it was always the same for each image and CD31 expression areas were measured automatically. TUNEL positive and CD31-positive cells were counted manually. For blinded quantification, images were organized in folder identified by letters by one person and quantified by another.