Nile blue chloride

Oxygen levels were measured using fluorophore-based microparticle sensors^{40 (link)}. Briefly, 2 g of 10–14 μm grade 7 silica gel (Sigma Aldrich) were stirred with 40 mL of 0.1 N NaOH for 30 minutes; then with 10 mL ethanol solutions of 0.5 mM tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride (Thermo Fisher) and 0.5 mM Nile blue chloride (Sigma Aldrich), respectively, for 30 minutes. The solution was then centrifuged for 20 minutes at 1900 × g. The pellet was washed and centrifuged with the same settings thrice with deionized water, and once with ethanol. The fluorophore-immobilized silica gel pellet was then dried in a 70 °C oven overnight. Simultaneously, a lid structure that fits the diffusion barrier pillar was milled with polycarbonate and silanized with trichloro(1 H, 1 H,2 H,2H-perfluorooctyle)silane (Sigma Aldrich) overnight. The following day, fluorophore-immobilized silica gel was mixed with PDMS of 1:10 base to curing agent (Sylgard 184 elastomer kit; Dow Corning) at a 1:20 ratio in an AR-100 Thinky mixer (Thinky U.S.A., Inc.). The mixture was then poured onto the microdevice’s pillar, covered with the lid, and cured overnight. Upon detaching the lid, the coated cap was imaged in 1x PBS equilibrated with normoxic air and then incubated with micropatterned cells. After 24 hours, fluorescence from the pillar surface was imaged.