Measurement of oxygen concentration within spheroids
For oxygen characterization, RuDPP was added to cell suspensions (2 µg.ml-1) before spheroid formation. After 5 days of culture the spheroids were live imaged using a laser scanning confocal microscope (Olympus FV1000) to capture Z-stack images throughout the spheroids. µ-Slide 8 well glass bottom slides from ibidi (cat# 80827) were used to image spheroids (excitation: 455nm, emission: 610nm). The imaging was performed by taking z-stacks at 10 µm intervals using 4X magnification. The fluorescent signals for spheroids cultured with/without PFC-MPs were quantitatively and spatially assessed using ImageJ (National Institutes of Health, Bethesda, MD, USA). To correlate fluorescent intensity to concentration of oxygen, a calibration curve was generated for RuDPP relating fluorescent intensity versus various oxygen concentrations (%O2).