Assay for measurement of intracellular sodium concentration ([Na+]i) and basolateral Na+/K+ ATPase activity in nonperfused isolated tubules or structures using the fluorescent indicator CoroNa Green. (Version 1.0)
Version
1.0
Notice
This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.
Authors
Rolando Carrisoza-Gaytan
Keywords
[‘kidney’, ‘organoid’, ‘mouse’, ‘hiPSC’, ‘nephron’, ‘imaging’]
Subjects
[‘Cell biology’, ‘Imaging’]
Release Date
2022-06-29
Abstract
This protocol describes methods for measurement of intracellular Na+ concentration ([Na+]i) and the assessment of basolateral Na+ exit through the Na+/K+ ATPase in tubular cells using a fluorescent probe.
Reagents
- Cell-Tak™ Cell and Tissue Adhesive (Corning®, cat # 354240): Prepare according to the manufacturer’s instructions.
- Poly-D-lysine hydrobromide mol wt 70000-150000, lyophilized powder (Millipore Sigma, cat# P6407): Prepare stock solution of 0.1% w/v and diluted working solutions of 0.01% (store at -20°C).
- CoroNa Green-AM (ThermoFisher Scientific; Cat# C36676): Prepare a 380 µM stock in DMSO (store 30 μL aliquots at -20°C) and then working 10 μM aliquots in Ringer’s solution (prepare 3 aliquots of 1 ml each for 3 sequential 10 min loads) before each experiment.
- Ouabain, Octahydrate – CAS 11018-89-6- Calbiochem (Millipore Sigma, Cat# 4995): Prepare a 20 mM w/v stock solution in H2O (use hot H2O) and store at 4°C. Prepare 2 mL of a 2 mM working solution in Ringer’s solution before each experiment.
- Solutions:
Equipment
Digital Fluorometry is performed in CoroNa Green-loaded cells or tubules, visualized using a Nikon S Fluor X40 objective (numeric aperture 0.9, working distance 0.3) attached to an inverted epifluorescence microscope (Nikon Eclipse Ti U) and excited at 490 nm using a wavelength switcher (DG-4 or LAMBDA Sutter) and images of the fluorescence emission at 530 nm are acquired at intervals of 3 to 5 sec using a Zyla 4.2 sCMOS camera (ANDOR Technology), interfaced with a digital imaging system (MetaFluor, Universal Imaging).
Procedure
- Single tubules are isolated from organoids by manual microdissection (on a cold stage under a stereoscopic microscope) or by collagenase digestion (L. Oxburgh protocol).
- Isolated tubules are transferred and affixed to a coverslip (No. 1 thickness) previously painted with a circular droplet (1 µL) of 0.01% poly-D-lysine (in H2O) or Cell-Tak. See below for representative photos of our chamber.
- Tubules are equilibrated in Ringer’s solution at 37°C for 45 min and the bathing solution exchanged at a rate of 10 mL/h using a peristaltic syringe pump.
- After equilibration, tubules are incubated with 10 µM of the acetoxymethyl ester (- AM) of CoroNa Green (ThermoFisher; in Ringer’s) added to the bath for 30 min in the absence of flow (bath containing fluorescent probe is exchanged every 10 min to minimize evaporative losses), at room temperature, and then rinsed for 10 min with Ringer’s solution at 37°C.
- Fluorometry is performed in CoroNa Green-loaded cells or tubules, visualized using a Nikon S Fluor X40 objective (numeric aperture 0.9, working distance 0.3) and excited at 490 nm, using a wavelength switcher (DG-4 or LAMBDA Sutter) and images of the fluorescence emission at 530 nm are acquired at intervals ranging from 30-60s using a Zyla 4.2 sCMOS camera (ANDOR Technology), interfaced with a digital imaging system (MetaFluor, Universal Imaging)
- Once a stable reading of the fluorescence intensity (FI) is observed (generally after 5 – 10 mins), agonists or antagonists of ion channels, transporters, can be added to the bath and FIs followed (for example, the Na+/K+ ATPase inhibitor ouabain, see anticipated results).
- The magnitude of changes in the FI in response to agonist or antagonist reflects changes in [Na+]i
Anticipated_Results
References
Fujishima A, Takahashi K, Goto M, Hirakawa T, Iwasawa T, Togashi K, Maeda E, Shirasawa H, Miura H, Sato W, Kumazawa Y, Terada Y. Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators. PLoS One. 16(1):e0246337, 2021 PMID: 33513193
Consortium
(Re)Building a Kidney (RBK) Consortium
