Lcachn 20xipc

All stained MSC samples were imaged using an inverted microscope (Etaluma LS720, Lumaview 720/600-Series software) with a 20 $\times$ phase contrast objective (Olympus, LCACHN 20XIPC) that allowed the acquisition of both phase contrast and fluorescence images. Approximately 600–900 images for each channel (i.e., phase contrast, 405 nm, 488 nm, and 594 nm) were obtained with a field of view $\sim 380\mathrm{\mu }$ m  $\times 380\mathrm{\mu }$ m. To conduct the time-lapse experiment, the same microscope and software was used in an incubator to image MSCs every 2 min over a period of 48 h (Videos S1, S2). While all of our selected antibodies have been previously validated, we also examined the non-specific binding by measuring the fluorescent intensity in samples that were only stained with secondary antibodies. Prior to further analyses, the background of the fluorescent data was evaluated and subtracted, following typical quantitative immunofluorescent microscopy procedures.