Assay for cell pH (pHi) and basolateral Na+/H+ exchange in nonperfused tubules using BCECF (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
[‘nephron’, ‘imaging’, ‘kidney’, ‘organoid’, ‘mouse’, ‘proximal tubule epithelial cells’, ‘hiPSC’]
Subjects
[‘Imaging’, ‘Cell biology’]
Release Date
2022-04-20
Abstract
This assay describes methods for measurement of intracellular pH and the assessment of basolateral Na+/H+ exchange activity in tubular cells.
Reagents
- Corning® Cell-Tak™ Cell and Tissue Adhesive, (prepare according provider instructions; Cat # 354240)
- Poly-D-lysine hydrobromide mol wt 70000-150000, lyophilized powder (Millipore sigma, Cat# P6407) Prepare stock solution 0.1% w/v and diluted working solutions 0.01% (store at -20°C)
- BCECF-AM (molecular Probes, Invitrogen) Stock solution in DMSO 1mg/mL (1.6mM; store at -20°C), prepare 20μM aliquots (3 x 1mL) before use.
- Solutions:
Equipment
Digital ratio fluorometry is performed in BCECF-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 alternately excited at 490 nm and 440 nm, using a wavelength switcher (DG-4 or LAMBDA Sutter) and images of the fluorescence emission at 530nm are acquired at intervals of 10 to 60 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 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 (1µL) of 0.01% poly-D-lysine (in H2O) or Cell-Tack. See figure1 and figure2 for representative photos of our chamber.
- Tubules are equilibrated in Na-Ringer’s solution at 37°C with bath exchanged continuously at a rate of 10mL/h using a peristaltic pump.
- After equilibration, tubules are incubated with 20 µM BCECF-acetoxymethyl ester (BCECF-AM; Molecular Probes) added to the bath of Na-Ringer’s for 30 min in the absence of flow (bath containing fluorescent probe is exchanged every 10 min to minimize evaporative losses) and then rinsed for 10 min with Na-Ringer’s solution.
- Digital ratio fluorometry is performed in BCECF-loaded cells or tubules, visualized using a Nikon S Fluor X40 objective (numeric aperture 0.9, working distance 0.3) and alternately excited at 490 and 440 nm, using a wavelength switcher (DG-4 or LAMBDA Sutter) and images of the fluorescence emission at 530nm are acquired at intervals of 10 to 60 sec using a Zyla 4.2 sCMOS camera (ANDOR Technology), interfaced with a digital imaging system (MetaFluor, Universal Imaging)
- Once stable fluorescence intensity ratios (FIRs; 490 nm/440 nm) are observed (generally after 5 – 10 mins), Na-Ringer’s solution in the bath is replaced by 0Na-Ringer’s solution (Na replaced by NMDG).
- Once stable FIRs are observed, Na-Ringer’s solution is restored to the bath.
- Finally, an intracellular calibration with three high-K Nigericin (10 µM) solutions, adjusted to pH values of 6.8, 7.3 and 7.8, is performed to convert FIRs into pHi values, based on the slope and y-intercept of the calibration curve, obtained by plotting FIR for each of the known pH solutions.
Figure1
Figure2
Anticipated_Results
Effect of basolateral Na on pHi in organoid cells
References
Liu W., Pastor-Soler N.M., Schreck C., Zavilowitz B., Kleyman T.R., and Satlin L.M. Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD). Am J Physiol Renal Physiol 302:F205-15, 2012. Epub 2011 Sep 28. PMID: 21957178 PMCID: PMC3251342
Consortium
(Re)Building a Kidney (RBK) Consortium
