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Cells were then washed twice with mitochondrial assay solution (220 mM mannitol [Sigma-Aldrich, M4125], 70 mM sucrose [Sigma-Aldrich, S7903], 10 mM KH 2 PO 4 [VWR, 470302], 2 mM HEPES [Fisher Scientific, BP310], and 1 mM EGTA [VWR, 97062]) and then overlaid with 175 L of mitochondrial assay solution supplemented with 10 mM sodium pyruvate (Sigma-Aldrich, P5280), 1 mM malate (Sigma-Aldrich, M0875), 4 mM ADP (Sigma-Aldrich, A5285), and Seahorse Plasma Membrane Permeabilizer (Agilent, 102504-100)

25 Prior to the pandemic, fentanyl mainly affected urban areas in the eastern regions of the U.S