Orm 10103

Control solution contained (mM): 118.5 NaCl, 4.7 KCl, 1.3 CaCl₂, 3.1 MgCl₂, 25 NaHCO₃, 2 NaH₂PO₄, and 5.5 d-glucose. Solutions containing different K⁺ concentrations were prepared by adding the appropriate amount of KCl. Solutions containing ouabain, an NKA inhibitor (Sigma-Aldrich), were prepared by dissolving ouabain directly in the control solution. Solutions containing 2-[(3,4-dihydro-2-phenyl-2H-1-benzopyran-6-yl)oxy]-5-nitro-pyridine (ORM-10103; an inhibitor of the NCX; Sigma-Aldrich) were prepared by first dissolving ORM-10103 in DMSO before it was added to the control solution. For the experiments involving ORM-10103, the final DMSO concentration was 0.1% (vol/vol) including the control solution. Solutions were continuously bubbled with 95% O₂–5% CO₂ to maintain a pH of 7.4. Experimental temperature was 37°C. Total flow of solutions in the muscle chamber was 15 ml/min being split just above and below the muscle to prevent any buildup of reactive oxygen species, which is quite large at 37°C (Edwards et al., 2007 (link)).

Ca²⁺ concentrations were assayed using the ratiometric intracellular
calcium indicator Fluo-4AM (Dojindo). DRG neurons cultured for 18 days were
loaded at room temperature for 30 min with 2 μM Fluo-4AM in Hank's balanced salt
solution (HBSS) containing the following (in mM): 140 NaCl, 3 KCl, 1
MgCl₂, 1 CaCl₂, and 10 HEPES, pH 7.3, along with 0.02%
Pluronic (Sigma). Neuronal cultures were illuminated with ordinary light to
reveal the neurons that were cultured with TNF-α and LPS overnight. After that,
we use saline or 1.4 μM KB-R7943(Tocris, UK) or 960 nM ORM-10103(Sigma, USA) to
incubate these neurons for 2 h. Neuronal cell bodies and neurites identified
from the visible light observation were selected for Ca²⁺ imaging.
Neurons were illuminated with 488 nm light using Laser confocal microscope
(OLYMPUS FV1000).