In order to derive Na+in from ratios of SBFI-labeled E10 motoneurons, ouabain (100µM, Na+/K+-ATPase inhibitor, Sigma), gramicidin (3µM, K+/Na+ ionophore, Invitrogen), and monensin (10µM, Na+/H+ antiporter, Sigma) were added to the bath solution in order to equilibrate intracellular and extracellular Na+, K+, and pH. Although we cannot rule out any effect of pH on SBFI ratios when Na+in is high (Diarra et al. 2001 (link)), we did not see significant differences in ratios when pH was changed from 6.8 to 8 in extracellular solutions containing ionophores and 75mM Na+ in an E10 cord (10 cells - pH of 6.8, 7.2, 7.4, 7.7, and 8, ANOVA p=0.75) or an E12 cord (7 cells - pH 7.4, 7.6, 7.8, 8, ANOVA p=0.66). For calibration, ratios were taken in solutions with various concentrations of Na+ (in mM: 155, 90, 75, 60, and 0) where the concentrations of K+ and Na+ ions summed to 155mM and Cl− = 50mM (Rose 1997 (link)). Ratios were then converted to Na+ concentrations based on the ratio values obtained in different solutions as described in the results.
Na k atpase inhibitor
Manufactured by Merck Group
The Na+/K+-ATPase inhibitor is a laboratory product used to inhibit the activity of the sodium-potassium ATPase enzyme. This enzyme is responsible for maintaining the electrochemical gradient across the cell membrane. The inhibitor can be used in various research applications to study the role of the Na+/K+-ATPase in cellular processes.
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2 protocols using na k atpase inhibitor
1
Measuring Intracellular Sodium in Motoneurons
2
Cytoskeleton and Ion Pump Perturbations
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To disrupt ion and fluid pumping, spheroids were treated with ouabain, a Na+/K+ ATPase inhibitor (Sigma) or 1-deamino-8-d -arginine vasopressin (ddAVP, Sigma), a vasopressin receptor agonist. Each was dissolved in distilled water at 1000× final concentration and diluted in cell culture media immediately before treatment for 4–24 h before DIC imaging. ouabain was used at a final concentration of 333 µM while ddAVP was used at a final concentration of 10 µM. For cytoskeletal inhibition (and controls) experiments, data were collected on at least five separate days from distinct samples. For fluid pumping inhibition experiments, data were collected on two separate days from distinct samples.
To perturb the actomyosin and microtubule cytoskeletons, cells were treated with a cytoskeletal inhibitor cocktail composed of latrunculin A (Sigma, 1 µM), Rho-kinase inhibitor Y-27632 (STEMCELL Technologies, 20 µM), MLCK inhibitor ML-7 (Enzo, 20 µM), and nocodazole (Sigma, 50 µM). The cytoskeletal inhibitor cocktail was made at 5× final concentration in L15 media and 100 µL were added to imaging well with 400 µL of L15.
To perturb the actomyosin and microtubule cytoskeletons, cells were treated with a cytoskeletal inhibitor cocktail composed of latrunculin A (Sigma, 1 µM), Rho-kinase inhibitor Y-27632 (STEMCELL Technologies, 20 µM), MLCK inhibitor ML-7 (Enzo, 20 µM), and nocodazole (Sigma, 50 µM). The cytoskeletal inhibitor cocktail was made at 5× final concentration in L15 media and 100 µL were added to imaging well with 400 µL of L15.
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