Rats were fasted overnight and anesthetized using 2.5% isoflurane in 1.5 l/min oxygen. A midline abdominal incision was made, and a single loop of mesentery was pulled out and placed in a customized heated (37°C) chamber filled with HEPES-PSS consisting of the following (in millimole per liter): 119 NaCl, 4.7 KCl, 1.17 MgSO4, 1.6 CaCl2, 24 NaHCO3, 0.026 EDTA, 1.17 NaH2PO4, 5.5 glucose, and 5.8 HEPES; pH was titrated to 7.4 using NaOH. A single LV was visualized, and increasing concentrations of cromakalim or minoxidil sulfate (0.01–10 μmol/l; log units) were added to the superfusate bathing the mesentery for 15 minutes at each drug concentration. Customized software developed in collaboration with our institution’s Nanomedicine Center was used to track individual lymph cells in flow as described earlier in detail (Sarimollaoglu et al., 2018; Stolarz et al., 2019). Using these data files, flow velocity and positive volumetric flow were calculated to evaluate the effect of KCOs on lymph flow in vivo.

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