Metamorph advanced imaging acquisition software v 7

Manufactured by Molecular Devices

MetaMorph Advanced Imaging acquisition software v.7.7.8.0 is a software application designed for image acquisition, processing, and analysis. The core function of the software is to capture, manage, and analyze digital images from a variety of microscopy and imaging systems.

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Lab products found in correlation

Ixon emccd camera, by Oxford Instruments (4 mentions) Csu x1 scanner, by Yokogawa (4 mentions) Ix81 inverted microscope, by Olympus (4 mentions) Fura 2 am, by Thermo Fisher Scientific (1 mentions) Spinning disk confocal microscope, by Olympus (1 mentions) Ixon electron multiplying charge coupled device camera, by Oxford Instruments (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Lysotracker, by Thermo Fisher Scientific (1 mentions) Planapon, by Olympus (1 mentions) Dq red bsa, by Thermo Fisher Scientific (1 mentions) Fluoromount g, by Southern Biotech (1 mentions)

7 protocols using metamorph advanced imaging acquisition software v 7

Intracellular Calcium Imaging Protocols

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Fura-2 Ca²⁺ imaging was carried out in cells loaded with 5 μM Fura-2 AM (Invitrogen) at 37 °C for 1 h, as described previously23 (link). In brief, fluorescence, at two excitation wavelengths, F₃₄₀ and F_380, was recorded with an EasyRatioPro system (PTI). Fura-2 ratios (F₃₄₀/F₃₈₀) were used to monitor changes in intracellular [Ca²⁺]. Lysosomal Ca²⁺ release was measured under a zero-Ca²⁺ external solution, which contained 145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA and 20 mM HEPES (pH 7.4); free [Ca²⁺]_o<10 nM (estimated with Maxchelator software http://maxchelator.stanford.edu/).
GCaMP imaging was performed in HeLa cells transfected with GCaMP7–TRPML1, a lysosome-targeted genetically-encoded Ca²⁺ sensor23 (link). The fluorescence intensity at 488 nm (F488) was recorded at 37 °C with the spinning-disk confocal live-imaging system, which included an Olympus IX81 inverted microscope, a × 60 or × 100 objective (Olympus), a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor) and MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices).

Zhang X., Cheng X., Yu L., Yang J., Calvo R., Patnaik S., Hu X., Gao Q., Yang M., Lawas M., Delling M., Marugan J., Ferrer M, & Xu H. (2016). MCOLN1 is a ROS sensor in lysosomes that regulates autophagy. Nature Communications, 7, 12109.

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Lysosomal Ca2+ Dynamics Imaging

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Time-lapse imaging was conducted on an Olympus spinning-disk confocal microscope equipped with a temperature controller. To detect Ca²⁺ release from lysosomes and TVs, we used GCaMP3-ML1-expressing ML1^PC parietal cells. GCaMP3, a single-wavelength genetically-encoded Ca²⁺ indicator was engineered using GFP and calmodulin (Tian et al., 2009 (link)), was fused directly to the cytoplasmic N-terminus of ML1 (Fig. S3F; detailed characterization see ref. (Shen et al., 2012 )). Upon Ca²⁺ release from TV stores, GCaMP3-ML1 fluorescence increased, which was monitored at an excitation wavelength of 480 nm (F₄₈₀) by an EasyRatio Pro imaging system (PTI). Cells were bathed in Tyrode’s solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Glucose, and 20 mM HEPES (pH 7.4). TV Ca²⁺ release was monitored in a zero Ca²⁺ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA, and 20 mM HEPES (pH 7.4). Free Ca²⁺ concentration was estimated by Maxchelator software (http://maxchelator.stanford.edu/). Images were analyzed with MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices) and Image J (NIH).

Sahoo N., Gu M., Zhang X., Raval N., Yang J., Bekier M., Calvo R., Patnaik S., Wang W., King G., Samie M., Gao Q., Sahoo S., Sundaresan S., Keeley T.M., Wang Y., Marugan J., Ferrer M., Samuelson L.C., Merchant J.L, & Xu H. (2017). Gastric acid secretion from parietal cells is mediated by a Ca2+ efflux channel in the tubulovesicle. Developmental cell, 41(3), 262-273.e6.

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Immunostaining of Muscle Tissue and Cells

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Muscle tissues, harvested and frozen in 2-methylbutane, prechilled in liquid nitrogen, were cryosectioned at 12 μm. After being washed with tris-buffered saline (TBS) + 0.025% Triton X-100, sections were blocked with 10% serum and 1% bovine serum albumin in TBS at room temperature for 2 hours. Cell lines and isolated primary cells cultured on coverslips were washed with PBS, fixed in 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 in PBS, and blocked in 1% bovine serum albumin in PBS. Fixed cells and cryosections were then incubated at 4°C overnight with primary antibodies targeting ML1, GFP, Lamp1, TFEB, dystrophin, or CD11b (M1/70.15.11.5.2, DSHB) at 1:50 or 1:200 solutions. Alexa Fluor secondary antibodies (Invitrogen) were then applied for 1 hour in the dark at room temperature, followed by 4′,6-diamidino-2-phenylindole counterstaining if necessary. Cells and tissue sections were imaged on a spinning disc confocal imaging system composed of an Olympus IX81 inverted microscope; 10×, 20×, and 60× Olympus objectives; a CSU-X1 scanner (Yokogawa); an iXon electron multiplying charge-coupled device camera (Andor); and MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices). Image analysis results were quantified in MetaMorph software.

Yu L., Zhang X., Yang Y., Li D., Tang K., Zhao Z., He W., Wang C., Sahoo N., Converso-Baran K., Davis C.S., Brooks S.V., Bigot A., Calvo R., Martinez N.J., Southall N., Hu X., Marugan J., Ferrer M, & Xu H. (2020). Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models. Science Advances, 6(6), eaaz2736.

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Monitoring Lysosomal Calcium Dynamics

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GCaMP3 Ca²⁺ imaging was performed in HEK cells that stably express GCaMP3-ML1, which are lysosome-targeted genetically encoded Ca²⁺ sensors³⁶. The fluorescence intensity at 488 nm (F₄₈₈) was recorded with the spinning disk confocal imaging system, which consisted of an Olympus IX81 inverted microscope, a 60X or 100X objective (Olympus), a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor), and MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices). Live imaging of ML1-GCaMP3 was performed on a heated stage.

Medina D.L., Di Paola S., Peluso I., Armani A., De Stefani D., Venditti R., Montefusco S., Scotto-Rosato A., Prezioso C., Forrester A., Settembre C., Wang W., Gao Q., Xu H., Sandri M., Rizzuto R., De Matteis M.A, & Ballabio A. (2015). Lysosomal calcium signaling regulates autophagy via calcineurin and TFEB. Nature cell biology, 17(3), 288-299.

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Monitoring Lysosomal Calcium Dynamics

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Live Cell Imaging Using Spinning Disc Confocal

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Live imaging of cells was performed on a heated and humidified stage using a Spinning Disc Confocal Imaging System. The system includes an Olympus IX81 inverted microscope, a 100X Oil objective NA 1.49 (Olympus, UAPON100XOTIRF), a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor). MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices) was used to acquire and analyze all images. LysoTracker (50 nM; Invitrogen) was dissolved in culture medium and loaded into cells for 30 min before imaging. MitoTracker was dissolved in culture medium and loaded into cells for 15 min before imaging (25 nM). Coverslips were washed 3 times with Tyrode’s and imaged in Tyrode’s.

Garrity A.G., Wang W., Collier C.M., Levey S.A., Gao Q, & Xu H. (2016). The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes. eLife, 5, e15887.

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Lysosomal Proteolytic Degradation Evaluation

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DQ-red-BSA was used as an artificial substrate to evaluate lysosomal proteolytic degradation (Yue et al., 2013 (link)). In brief, cells were treated with DQ-red-BSA (10 µg/ml; Thermo Fisher Scientific) for 2 h at 37°C in complete culture medium. After removal of extracellular DQ-red-BSA by washing twice with PBS, cells were starved in amino acid– and serum-free DF12 medium (US Biological) for 2 h to trigger autophagic degradation. To examine the effects of BK blockers on lysosomal proteolytic activities, paxilline (10 µM), quinidine (500 µM), or IBTX (100 nM) was applied during starvation. Cells were then fixed with 4% PFA for 15 min at RT, washed twice with PBS, and mounted on slides with Fluoromount-G (SouthernBiotech). DQ-red-BSA fluorescence was detected with a 561/607 filter set using the Spinning Disc Confocal Imaging System under an 60× oil objective NA 1.42 (Olympus; PlanApo N). Images were acquired and analyzed with MetaMorph Advanced Imaging acquisition software v. 7.7.8.0 (Molecular Devices). Quantification was performed using ImageJ.

Wang W., Zhang X., Gao Q., Lawas M., Yu L., Cheng X., Gu M., Sahoo N., Li X., Li P., Ireland S., Meredith A, & Xu H. (2017). A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores. The Journal of Cell Biology, 216(6), 1715-1730.

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