Quantifying Interstitial Fluid Drainage in Mice

Drainage of interstitial fluids was quantified using Evans Blue injections in the footpad of WT and Cx43^+/− mice, as described previously (Meens et al., 2017 (link)). In brief, mice were anesthetized and injected with 5 μl 5% Evans Blue (dissolved in PBS) in the left footpad using a micro syringe. After 15 min, blood was collected by puncturing the left ventricle and centrifuged 15 min at 5,000 rpm (4°C). Formamide (Sigma-Aldrich, St. Louis, MO) was added to each serum sample (500 μL Formamide/200 μL serum) and the mix was incubated overnight at 55°C. Thereafter, presence of Evans Blue in the serum was quantified by measuring the fluorescence using a SpectraMax Paradigm Multi-Mode Microplate reader (excitation: 620 nm; emission 670 nm; Molecular Devices, Sunnyvale, CA).