Lysosomal pH was measured as described previously (Guha, et al., 2013 (link)). In brief, differentiated Luhmes cells were grown in black 96-well plates, rinsed 3 times with isotonic solution [105 mM NaCl, 5 mM KCl, 6 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 mM Na HEPES, 5 mM NaHCO3, 60 mM mannitol, 5 mM glucose, 0.5 mM MgCl2, and 1.3 mM CaCl2], then incubated with 5 μM LysoSensor Yellow/Blue (Invitrogen, L7545) for 3 min. 15 min post-incubation, fluorescence was measured on a SpectraMax plate reader (Molecular device). Lysosomal pH was determined from the ratio of emission at 450 versus 510 nm (365 nM excitation) and calibrated by exposing cells to 20 μM H+/K+ ionophore nigericin in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES), 110 mM KCl, and 20 mM NaCl (pH 4.0–6.0) for 15 min. Comparisons were always made between measurements from the same plate and normalized for each plate.
Lysosensor yellow blue
Manufactured by Thermo Fisher Scientific
LysoSensor Yellow/Blue is a fluorescent probe that can be used to detect and image acidic organelles, such as lysosomes, in living cells. The probe exhibits a pH-dependent shift in fluorescence emission, allowing for ratiometric detection of changes in lysosomal pH.
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Lab products found in correlation
Prism 7, by GraphPad (2 mentions)
Lysotracker red, by Thermo Fisher Scientific (2 mentions)
Spectramax plate reader, by Molecular Devices (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
96 well black wall black bottom plates, by Thermo Fisher Scientific (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
Foetal calf serum, by GE Healthcare (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Mito tempo, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
8 protocols using lysosensor yellow blue
1
Measuring Lysosomal pH in Differentiated Luhmes Cells
2
Lysosomal pH Analysis in Parkinson's Disease
3
Lysosomal pH Measurement in ATII Cells
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ATII cells were incubated with 100 nM LysoSensor Yellow/Blue (Invitrogen) at 37°C for 30 min. Cells were washed with experimental bath solution and imaged on the Zeiss Observer microscope (dichroic filter 400 nm, emission filter 490–530 nm) and 40× fluar oil objective using Metafluor software. After background subtraction, LB intensities at 340 and 380 nm excitation were measured in ImageJ and the 340/380 nm ratio was used for pH calculation.
Calibration of the ratio to pH relation was done as described previously [51] by a free solution calibration. Bath solution with 1 µM LysoSensor was adjusted to pH values from 3 to 8 and imaged as described. The calibration curve could be fitted well with pH = c−log10((a−b)/(ratio−b)−1), where a, b and c are constants.
Calibration of the ratio to pH relation was done as described previously [51] by a free solution calibration. Bath solution with 1 µM LysoSensor was adjusted to pH values from 3 to 8 and imaged as described. The calibration curve could be fitted well with pH = c−log10((a−b)/(ratio−b)−1), where a, b and c are constants.
4
Lysosomal pH Analysis in Astrocytes
5
Ratiometric Lysosomal pH Analysis
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For lysosomal pH analysis, the ratiometric dye LysoSensor Yellow/Blue (Invitrogen) was used. Neurons were plated at 10,000 cells per well on 96-well black-wall black-bottom plates (Thermo Scientific), labeled with dye (1 μM) for 10 min prior to rinsing 2x with HBSS buffer. Cells were imaged using a Synergy H1 hybrid reader (Bio-Tek; reading at excitation 329/384, emission 440/540). Then, cells were incubated for 5 min at 37°C with pH calibration standards (pH of 3.96, 4.46, 4.96, 5.47, and 5.97) prepared in 20 mM 2-(N-morpholino)ethanesulfonic acid, 110 mM KCl, and 20 mM NaCl freshly supplemented with 30 μM nigericin and 15 μM monensin. A pH standard curve was determined for each genotype using GraphPad Prism 7 and individual baseline pH values were interpolated from these standard curves.
6
Lysosomal Imaging and Trafficking
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Materials were obtained from Sigma unless otherwise indicated. Tissue culture media and supplements were from Gibco. Foetal Calf Serum (FCS) was from PAA laboratories. LysoSensor yellow/blue, BODIPY-LacCer and LysoTracker red were from Invitrogen. U18666A was from Affinity Research
7
Cardiomyocyte Metabolic Function and Viability
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General chemicals and reagents were obtained from Sigma unless otherwise specified. Mito-TEMPO was from Santa Cruz Biotechnology. Low-glucose DMEM with phenol red or without phenol red, TMRE, LysoTracker Red, LysoSensor Blue/Yellow, DCFDA and Laminin were acquired from Invitrogen. Rapamycin was from Cell Signaling Technology. Trypsin and Collagenase were purchased from Willington. Antibodies were obtained from Santa Cruz Biotechnology (TFEB), Cell signaling Technology (total and phosphoryla-ted S6 Kinase, COX4), MBL (LC3), R&D Systems (GAPDH), Sigma (β-actin), Abcam (active caspase 3) and Abnovo (p62). Secondary antibodies for immunohistochemistry (donkey anti-mouse antibody-Alexa Fluor 555 and donkey anti-rabbit antibody-Alexa Fluor 488) were from Invitrogen. Adenovirus purification kit, ATPlite kit and TUNEL kit were purchased from Adenopure, Perkin Elmer and Roche, respectively. Full-length human TFEB-GFP adenovirus was purchased from Vector BioLabs, and adeno-GFP virus was used as a control. MOIs of the two adenovirus were adjusted to infect the adult cardiomyocytes for achieving an equal expression level of GFP as described previously. Experiments were started 24 h following adenovirus infection. Contractile function and intracellular calcium measurements were performed 24 h following AL-LC exposure.
8
Lysosomal pH Measurement in Cardiomyocytes
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Following designated hour treatment with vehicle, Con-LC and AL-LC (20 μg/ml), cultured cardiomyocytes were incubated with cell permeable, lysosomal-specific probe LysoTracker (Invitrogen) at a concentration of 100 nM for 30 min. Cardiomyocytes were washed twice with warm PBS. Cell images were acquired using LSM700 confocal microscopy (excitation wavelength at 555 nm) and analyzed with ImageJ software. For determination of lysosomal pH, cardiomyocytes were incubated with LysoSensor Blue/Yellow (Invitrogen) at a concentration of 1 μM for 3 min prior to measurement using LSM710 two-photon confocal microscopy (excitation wavelength was 720 nm, and emission wavelengths were collected from 400 to 461 nm for the blue emission and 510 to 630 nm for the yellow emission). Images were analyzed using imageJ software. Green fluorescence signal (blue emission) represents basic conditions, and red fluorescence signal (yellow emission) represents acidic conditions. Pseudocoloring (the ratio of green/red) was done to represent lysosomal pH.
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