Csu x1 scanner, by Yokogawa - Product details

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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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Live Imaging and Immunofluorescence of Myoblasts

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Live imaging of C2C12 or primary myoblasts was performed on a heated stage on a 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). For immunofluorescence detection, cells or muscle sections were fixed with 4% PFA, permeabilized with 0.03% Triton X-100, and stained with various primary antibodies: EEA1 (Abcam), Lamp1 (ID4B), dystrophin, β-DG, integrin β1 (Iowa Hybridoma Bank), and laminin (Chemicon).

Cheng X., Zhang X., Gao Q., Samie M.A., Azar M., Tsang W.L., Dong L., Sahoo N., Li X., Zhuo Y., Garrity A.G., Wang X., Ferrer M., Dowling J., Xu L., Han R, & Xu H. (2014). An Intracellular Ca2+ Channel is Required For Sarcolemma Repair to Prevent Muscular Dystrophy. Nature medicine, 20(10), 1187-1192.

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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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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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Live Imaging and Immunofluorescence of Myoblasts

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Live imaging of C2C12 or primary myoblasts was performed on a heated stage on a 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). For immunofluorescence detection, cells or muscle sections were fixed with 4% PFA, permeabilized with 0.03% Triton X-100, and stained with various primary antibodies: EEA1 (Abcam), Lamp1 (ID4B), dystrophin, β-DG, integrin β1 (Iowa Hybridoma Bank), and laminin (Chemicon).

Cheng X., Zhang X., Gao Q., Samie M.A., Azar M., Tsang W.L., Dong L., Sahoo N., Li X., Zhuo Y., Garrity A.G., Wang X., Ferrer M., Dowling J., Xu L., Han R, & Xu H. (2014). An Intracellular Ca2+ Channel is Required For Sarcolemma Repair to Prevent Muscular Dystrophy. Nature medicine, 20(10), 1187-1192.

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Fluorescence and Time-Lapse Imaging of Cellular Dynamics

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Fluorescence and time-lapse imaging was conducted in a spinning-disk confocal imaging system composed of an Olympus IX81 inverted microscope, 10 ×, 20 ×, and 60 × Olympus objectives, a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor), a temperature controller, and MetaMorph Advanced Imaging acquisition software v.7.7.8.0. FluoZin-3-AM and LysoTracker were loaded according to the manufacturer’s instructions. Briefly, FluoZin-3AM (1 mM stock solution in DMSO) was diluted to a final concentration of 3 μM in respective culture mediums. A 1 mM stock solution of LysoTracker RED DND-99 was diluted to a final concentration of 0.5 μM in respective culture mediums. Cells were loaded with the dye-containing buffer for 30 min before image collection. For time-lapse imaging of PI fluorescence (Videos S1 and S2), cells were challenged with DMEM supplemented with 10 μg/mL PI, and images were taken at an interval of 5 or 10 min for M12 cells and MeWo cells, respectively. For MitoTracker live imaging, cells were loaded with MitoTracker and treated with ML-SAs (1h for melanocytes and MeWo cells, 30 min for M12 cells). For most inhibitor experiments, drugs were pretreated for 30 min. Images were acquired and analyzed with MetaMorph Advanced imaging acquisition software v.7.7.8.0 and ImageJ (NIH).

Du W., Gu M., Hu M., Pinchi P., Chen W., Ryan M., Nold T., Bannaga A, & Xu H. (2021). Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma. Cell reports, 37(3), 109848.

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Fluorescence and Time-Lapse Imaging of Cellular Dynamics

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Fluorescence and time-lapse imaging was conducted in a spinning-disk confocal imaging system composed of an Olympus IX81 inverted microscope, 10 ×, 20 ×, and 60 × Olympus objectives, a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor), a temperature controller, and MetaMorph Advanced Imaging acquisition software v.7.7.8.0. FluoZin-3-AM and LysoTracker were loaded according to the manufacturer’s instructions. Briefly, FluoZin-3AM (1 mM stock solution in DMSO) was diluted to a final concentration of 3 μM in respective culture mediums. A 1 mM stock solution of LysoTracker RED DND-99 was diluted to a final concentration of 0.5 μM in respective culture mediums. Cells were loaded with the dye-containing buffer for 30 min before image collection. For time-lapse imaging of PI fluorescence (Videos S1 and S2), cells were challenged with DMEM supplemented with 10 μg/mL PI, and images were taken at an interval of 5 or 10 min for M12 cells and MeWo cells, respectively. For MitoTracker live imaging, cells were loaded with MitoTracker and treated with ML-SAs (1h for melanocytes and MeWo cells, 30 min for M12 cells). For most inhibitor experiments, drugs were pretreated for 30 min. Images were acquired and analyzed with MetaMorph Advanced imaging acquisition software v.7.7.8.0 and ImageJ (NIH).

Du W., Gu M., Hu M., Pinchi P., Chen W., Ryan M., Nold T., Bannaga A, & Xu H. (2021). Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma. Cell reports, 37(3), 109848.

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Csu x1 scanner

Lab products found in correlation

8 protocols using csu x1 scanner

Intracellular Calcium Imaging Protocols

Live Imaging and Immunofluorescence of Myoblasts

Immunostaining of Muscle Tissue and Cells

Monitoring Lysosomal Calcium Dynamics

Monitoring Lysosomal Calcium Dynamics

Live Imaging and Immunofluorescence of Myoblasts

Fluorescence and Time-Lapse Imaging of Cellular Dynamics

Fluorescence and Time-Lapse Imaging of Cellular Dynamics

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