Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: The CellProfiler program processed fluorescence pictures of untreated fibroblast cells (0 µM H 2 O 2 ) through the pipeline for estimating mitochondrial H 2 O 2 levels. The fibroblast cells were co-stained with Hoechst 33342, MITO-ID® Red, and MitoPY1 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The Hoechst 33342 (blue) and MITO-ID® Red (red) channels were dedicated to identifying the Nuclei and cell's boundary outlines for the cytoplasm identification. B. The MitoPY1 channels' green-speckled fluorescence was used to recall hydrogen peroxide (H 2 O 2 ). C. Mitochondrial H 2 O 2 was analyzed by relating the green-speckled fluorescence to the cells identified from Hoechst 33342 and MITO-ID® Red channels. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: The CellProfiler program processed fluorescence pictures of fibroblast cells treated with 200 µM of H 2 O 2 for 1 h. The fibroblast cells were co-stained with Hoechst 33342, MITO-ID® Red, and MitoPY1 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The Hoechst 33342 (blue) and MITO-ID® Red (red) channels were dedicated to identifying the nuclei and cell's boundary outlines for the cytoplasm identification. B. The MitoPY1 channels' green-speckled fluorescence was used to recall hydrogen peroxide (H 2 O 2 ). C. Mitochondrial H 2 O 2 was analyzed by relating the green-speckled fluorescence to the cells identified from Hoechst 33342 and MITO-ID® Red channels. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: The CellProfiler program processed fluorescence pictures of fibroblast cells treated with 400 µM of H 2 O 2 for 1 h. The fibroblast cells were co-stained with Hoechst 33342, MITO-ID® Red, and MitoPY1 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The Hoechst 33342 (blue) and MITO-ID® Red (red) channels were dedicated to identifying the Nuclei and cell's boundary outlines for the cytoplasm identification. B. The MitoPY1 channels' green-speckled fluorescence was used to recall hydrogen peroxide (H 2 O 2 ). C. Mitochondrial H 2 O 2 was analyzed by relating the green-speckled fluorescence to the cells identified from Hoechst 33342 and MITO-ID® Red channels. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of untreated fibroblast cells (ethanol, 0 µM of FCCP) via the pipeline for measuring MMP levels. The fibroblast cells were co-stained with Hoechst 33342 and JC-10 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. J-monomers (green-fluorescence) from JC-10 Alexa Fluor 488 channels associated with nuclei were specified and segmented. B. J-aggregates (orange-fluorescence) indicating polarized MMP from the JC-10 Rhodamine channels were inferred. C. The areas of J-aggregates masked by J-monomers (middle image) representing orange-fluorescence and the J-monomers masked by J-aggregates (right image) representing green-fluorescence were identified to calculate the intensity by the Mask Object module. The MMP alterations can be evaluated quantitatively using the intensity ratio of orange-fluorescence aggregates to green-fluorescence monomers. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of fibroblast cells treated with 0.01 µM of FCCP for 1 h via the pipeline for measuring MMP levels. The fibroblast cells were co-stained with Hoechst 33342 and JC-10 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. J-monomers (green-fluorescence) from JC-10 Alexa Fluor 488 channels associated with nuclei were specified and segmented. B. J-aggregates (orange-fluorescence) indicating polarized MMP from the JC-10 Rhodamine channels were inferred. C. The areas of J-aggregates masked by J-monomers (middle image) representing orange-fluorescence and the J-monomers masked by J-aggregates (right image) representing green-fluorescence were identified to calculate the intensity by the Mask Object module. The MMP alterations can be evaluated quantitatively using the intensity ratio of orange-fluorescence aggregates to green-fluorescence monomers. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of fibroblast cells treated with 0.1 µM of FCCP for 1 h via the pipeline for measuring MMP levels. The fibroblast cells were co-stained with Hoechst 33342 and JC-10 fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. J-monomers (green-fluorescence) from JC-10 Alexa Fluor 488 channels associated with nuclei were specified and segmented. B. J-aggregates (orange-fluorescence) indicating polarized MMP from the JC-10 Rhodamine channels were inferred. C. The areas of J-aggregates masked by J-monomers (middle image) representing orange-fluorescence and the J-monomers masked by J-aggregates (right image) representing green-fluorescence were identified to calculate the intensity by the Mask Object module. The MMP alterations can be evaluated quantitatively using the intensity ratio of orange-fluorescence aggregates to green-fluorescence monomers. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of untreated fibroblast cells (ethanol, 0 µM of phenanthroline) through the pipeline for analyzing the levels of mitochondrial fragmentation and length. The fibroblast cells were co-stained with Hoechst 33342 and MITO-ID® Red fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The image from the MITO-ID® Red channel was altered to Grayscale and enhanced the red fluorescent intensity. B. Mitochondria were identified by tagging the enhanced red fluorescence using the Thresholding strategy. C. The Morph module was utilized to shrink mitochondria into the skeleton and mark the endpoints of the skeleton. D. The mean mitochondrial length and fragmentation per cell were estimated through the Measure Object Skeleton module by measuring the distance between the shrunken endpoints within the mitochondrial skeleton. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging

Journal: MethodsX

Article Title: Methods for mitochondrial health assessment by High Content Imaging System

doi: 10.1016/j.mex.2022.101685

Figure Lengend Snippet: CellProfiler program processed fluorescence pictures of fibroblast cells treated with 50 µM of phenanthroline for 4 through the pipeline for analyzing the levels of mitochondrial fragmentation and length. The fibroblast cells were co-stained with Hoechst 33342 and MITO-ID® Red fluorescent dyes. Imaging of stained cells was visualized by the Operetta CLS with the 40x NA objective lens. A. The image from the MITO-ID® Red channel was altered to Grayscale and enhanced the red fluorescent intensity. B. Mitochondria were identified by tagging the enhanced red fluorescence using the Thresholding strategy. C. The Morph module was utilized to shrink mitochondria into the skeleton and mark the endpoints of the skeleton. D. The mean mitochondrial length and fragmentation per cell were estimated through the Measure Object Skeleton module by measuring the distance between the shrunken endpoints within the mitochondrial skeleton. Scale bars represent 10 µm.

Article Snippet: The images of stained cells were visualized using the Operetta CLS TM High-Content Analysis System (PerkinElmer) in the confocal or non-confocal with two peaks of autofocus mode and a water objective lens, 40x high numerical aperture (NA), which had a high refractive index.

Techniques: Fluorescence, Staining, Imaging