We performed confocal imaging as described previously.31 (link) Colonies were transferred to a 96-well microtiter plate with 100 μL TP liquid medium in each well and then pre-cultured in air under 150 μmol photons m−2 s−1 on an orbital shaker. After ~16 hr of growth, 10 μL cells were transferred onto an μ-Slide 8-well glass-bottom plate (Ibidi) and 200 μL of 1% TP low-melting-point agarose at ~35 °C was overlaid to restrict cell movement. Cell samples were imaged using a Leica SP5 confocal microscope with the following settings: Venus, 514 nm excitation with 530/10 nm emission; and chlorophyll, 514 nm excitation with 685/40 nm emission. All confocal microscopy images were analyzed using Fiji.87 (link) For each strain, a confocal section through a cell showing the predominant localization pattern was captured and analyzed.
μ slide 8 well glass bottom plate
Manufactured by Ibidi
Sourced in Germany
The μ-Slide 8-well glass bottom plate is a high-quality cell culture plate designed for microscopy applications. It features eight individual wells with a glass bottom, providing a clear surface for high-resolution imaging. The plate is made from durable, biocompatible materials and is suitable for a variety of cell culture and imaging techniques.
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Lab products found in correlation
Lsm 800, by Zeiss (6 mentions)
Filipin, by Merck Group (2 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Tcs sp8 x confocal microscope, by Leica (1 mentions)
Las x software, by Leica (1 mentions)
Mitochondrial superoxide detection kit, by Abcam (1 mentions)
Fv1200, by Olympus (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Mito tracker red cmxros, by Beyotime (1 mentions)
Dcfh da ros assay kit, by Beyotime (1 mentions)
D galactose, by Merck Group (1 mentions)
Mitoview red, by GeneCopoeia (1 mentions)
Tcs sp8, by Leica (1 mentions)
Mdivi 1, by Merck Group (1 mentions)
Fluo 4 am calcium assay kit, by Beyotime (1 mentions)
Catalase, by Merck Group (1 mentions)
Tris hcl ph 8, by Merck Group (1 mentions)
Matlab, by MathWorks (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
405 nm laser, by Coherent Inc (1 mentions)
12 protocols using μ slide 8 well glass bottom plate
1
Confocal Imaging of Microalgae Cells
2
Hypoxia-Induced Mitochondrial Oxidative Stress
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hDASMCs were plated at an experimentally determined, optimal density for fluorescence imaging in a μ‐Slide 8 Well Glass Bottom plate (part number 80827; Ibidi, Fitchburg, WI). The next day, cells were loaded with 10 μmol/L of MitoROS 580 dye using the Mitochondrial Superoxide Detection Kit (ab219943; Abcam). MitoROS was incubated with the cells for 30 minutes at 37 °C in a hypoxic incubator, in the presence of either DMSO, 50 μmol/L S1QEL, 100 μmol/L S3QEL, or 10 μmol/L rotenone. After loading, cell chambers were placed in an OkoLab stage‐top microscope incubator, and imaging was performed using a Leica TCS SP8 X confocal microscope (excitation 540 nm, emission 570–720 nm, 2.5 frames/second, Leica Microsystems). Cells were allowed to equilibrate in hypoxia for 10 minutes, before imaging for 10 minutes in hypoxia followed by 10 minutes of normoxia. Images were obtained using LAS‐X software (Leica) and representative images made using Fiji (ImageJ). For each treatment group, ROI were drawn around cells to track changes in MitoROS fluorescence over time. To compare changes in MitoROS fluorescence induced by increased O2 content, ROI intensity over the last 4 images of normoxic incubation were compared with the initial 4 images taken during hypoxic incubation.
3
Monitoring polyIC Internalization and Endocytic Trafficking
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MDA-MB-468 (10000 cells/well) and MCF7 (7000 cells/well) cells were plated in a μ-Slide 8 Well Glass Bottom plate (Ibidi). 48 hours after plating, the medium was replaced with fresh medium containing 1μM sulforhodamine G (Biotium Inc.) and the plate was placed in the 37℃ chamber of a confocal microscope (FV-1200 Olympus, Japan). The cells were then treated with 1μg/ml Cy3-polyIC alone, or 1μg/ml Cy3-polyIC which had been pre-incubated with dsRBEC (polyIC:dsRBEC, weight:weight ratio of 1:2). PolyIC internalization was monitored for 2 hours. For endosomal localization we treated MDA-MB-468 cells with polyIC/dsRBEC (as described above) and 5μg/ml AlexaFluor 647-labeled transferrin (Jackson ImmunoResearch Laboratories, Inc) simultaneously.
4
Mitochondrial Membrane Potential in H9c2 Cells
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H9c2 cells were seeded into μ-Slide 8-well glass bottom plate (#80826, ibidi, Germany) at a total number of 1 × 104 per well. To measure the mitochondrial membrane potential (MMP), JC-1 mitochondrial membrane potential assay kit (Beyotime, China) was used according to the manufacturer's protocol. After treatments of MPDV (0, 10, 20, 40%), cells were incubated with 10 μg/ml JC-1 at 37°C for 20 min. The fluorescent images of JC-1 were captured by red and green fluorescence, respectively with Confocal Microscopy (LSM 800, ZEISS, USA) equipped with a 63× oil immersion objective. For the quantification of MMP, H9c2 cells were seeded in 96 well black/clear bottom plates. Ex 488/Em 535 nm and Ex 550/Em 600 nm were used and MMP was calculated by the ratio of red to green fluorescence.
5
Mitochondrial Morphology Visualization
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H9c2 cells were seeded into μ-Slide 8-well glass bottom plate (#80826, ibidi, Germany) at a total number of 10,000 per well. After treatments of MPDV (0, 10, 20, 40%), cells were incubated with 100 nM MitoTracker Red CMXRos (Beyotime, China) at 37°C for 20 min. Then cells were washed using Dulbecco's Modified Eagle Medium (DMEM) for five times. The images of mitochondrial morphology were captured by confocal microscopy (LSM 800, ZEISS, USA) equipped with a 63× oil immersion objective. Red fluorescence represented mitochondria stained by MitoTracker Red CMXRos.
6
ROS Measurement in H9c2 Cells
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H9c2 cells were seeded into μ-Slide 8-well glass bottom plate (#80826, ibidi, Germany) at a total number of 1 × 104 per well. To measure the Reactive Oxygen Species (ROS) level, DCFH-DA ROS assay kit (Beyotime, China) was used according to the manufacturer's protocol. After treatments of MPDV (0, 10, 20, 40%), cells were incubated with 5 μM DCFH-DA at 37°C for 30 min. The fluorescent images of DCFH-DA were captured by green fluorescence with Confocal Microscopy (LSM 800, ZEISS, USA) equipped with a 63× oil immersion objective.
7
Mitochondrial Morphology Analysis in H9c2 Cells
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H9c2 cells were seeded into a μ-Slide 8-well glass bottom plate (#80826, ibidi, Germany) at a total number of 7500 per well. After treatments of D-galactose (0, 10, 20, and 40 g/l, Sigma, USA) or Mdivi-1 (40 μM, Sigma, USA), the cells were incubated with 50 nM MitoView Red (GeneCopoeia, USA) at 37°C for 30 min. Then, the cells were washed with PBS for three times. Mitochondrial morphology in each group was captured using a confocal microscope (Leica TCS SP8, Germany) equipped with a 63x oil immersion objective. Red fluorescence represents the mitochondria stained by MitoView Red.
8
Measuring Calcium Levels in H9c2 Cells
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H9c2 cells were seeded into μ-Slide 8-well glass bottom plate (#80826, ibidi, Germany) at a total number of 1 × 104 per well. To measure the calcium (Ca2+) ion level, Fluo-4 AM calcium assay kit (Beyotime, China) was used according to the manufacturer's protocol. After treatments of MPDV (0, 10, 20, 40%), cells were incubated with 2 μM Fluo-4 AM probe at 37°C for 30 min. The fluorescent images of Fluo-4 AM were captured by green fluorescence with confocal microscopy (LSM 800, ZEISS, USA) equipped with a 63× oil immersion objective.
9
Quantifying Cellular Cholesterol Levels
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Following treatment with Cholesterol:Methyl-β-Cyclodextrin complexes, cellular cholesterol levels were quantified by filipin (Sigma) staining and confocal imaging. CHO K1 and U2OS cells were seeded in μ-Slide 8 Well Glass Bottom plates (ibidi). After 24 hours of incubation, cells were incubated for 1 hour with Cholesterol:Methyl-β-Cyclodextrin complexes. Cells were then rinsed three times with PBS and fixed with 3% PFA for 1 hour at room temperature. Afterwards, to quench PFA, cells were rinsed three times with PBS and incubated with 1.5 mg/mL glycine in PBS for 10 min at room temperature. A stock solution of filipin (25 mg/mL in DMSO) was prepared and diluted to prepare a working solution of 50 µg/mL in PBS, supplemented with 10% FCS. Cells were stained with filipin for 2 hours at room temperature and rinsed three times with PBS. Imaging was performed using a confocal laser scanning microscope (Zeiss LSM800) with a 63× oil objective.
10
Quantifying Cellular Cholesterol Levels
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Cellular cholesterol levels were quantified by filipin (Sigma-Aldrich) staining and confocal imaging. CHO K1 and U2OS cells were seeded in μ-Slide 8-well glass bottom plates (ibidi) 1 d before imaging. Cells were incubated for either 1 h with Cholesterol:Methyl-β-Cyclodextrin complexes or for 24 h with 5 μM mevastatin and 50 μM mevalonate in the presence of de-lipidized serum, in culture conditions. Cells were then rinsed three times with PBS and fixed with 3% PFA for 1 h at room temperature. Afterwards, to quench PFA, cells were rinsed three times with PBS and incubated with 1.5 mg/ml glycine in PBS for 10 min at room temperature. A stock solution of filipin (25 mg/ml in DMSO) was prepared and diluted to prepare a working solution of 50 μg/ml in PBS, supplemented with 10% FCS. Cells were stained with filipin for 2 h at room temperature and rinsed three times with PBS. Imaging was performed in PBS at room temperature using a confocal laser scanning microscope (LSM800; Zeiss) with a 63× 1.4 NA Oil DIC objective lens (Zeiss). Images were acquired using the software ZEN 2.6 (blue edition). In addition to filipin fluorescence, GFP fluorescence (coming from FGF2-GFP) was acquired as a reference of the cells. Confocal images were then analyzed through Fiji (Schindelin et al., 2012 (link)).
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