Zen 3.4 blue software

Semi-automated analysis was done using the ZEN 3.4 Blue software (Zeiss). Blastocysts and subcellular structures were manually masked. The ZEN 3.4 Blue inbuilt function zone of influence analysis was carried out on the masked images to determine the number of mitochondria surrounding each LD (radius of 80 pixels). In cases where LDs occurred as clusters, binary dilation with a count of 15 was used to fuse the LDs in a cluster.

The binding assay of melanoma cells to immobilised proteins under static conditions was performed as described before [17 (link)]. For each experiment, a sample in which we checked the lack of binding of melanoma cells to immobilized BSA served as an internal control [17 (link)]. Images were acquired for all samples with identical settings with an AxioObserver.Z1 inverted microscope using a Plan-Neofluar 10×/0.3 objective, an Axiocam 712 mono camera and the ZEN 3.4 blue software (Zeiss, Feldbach, Switzerland). The number of bound melanoma cells was quantified in 3 FOVs per sample using Fiji/ImageJ, and the bound cells per mm² were calculated as the average number of cells divided by the FOV area.