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).