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In carta image analysis software

Manufactured by GE Healthcare
Sourced in Australia

IN Carta image analysis software is a digital pathology tool that enables users to view, analyze, and manage digital slide images. The software provides a platform for image processing and quantitative analysis of microscopic samples.

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10 protocols using in carta image analysis software

1

Quantifying Cellular Acetyl-Tubulin Levels

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To measure cellular acetyl-tubulin levels, cells were seeded, treated, fixed, and permeabilized as described above. Tubastatin was used as a positive control [85 (link)]. After blocking, cells were exposed to mouse monoclonal anti-acetyl-tubulin antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). Average cellular acetyl-tubulin intensity was automatically quantified for each acquired images. Data were standardized over the non-treated control (100%) and expressed as mean ± SD of at least quadruplicates from one assay. At least 1 × 103 cells were analyzed for each treatment.
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2

Quantifying Oxidative Stress by Nitrotyrosine

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To assess if the test compounds induce oxidative damage, cells were seeded, treated, fixed, and permeabilized as described above. Shikonin was used as a positive control [86 (link)]. After blocking, cells were exposed to mouse monoclonal anti-3-nitrotyrosine antibody (1:500 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). Average cellular nitrotyrosine intensity was automatically quantified for each acquired images. Data were standardized over the non-treated control (100%) and expressed as mean ± SD of at least 8 replicates from one assay. At least 2 × 103 cells were analyzed for each treatment.
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3

Quantitative Analysis of Nuclear Morphology

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To quantitate nuclear morphology, 384-well plates (781091, µClear, Greiner, Ryde, NSW, Australia) were coated with rat tail collagen for 45 min (1:20 in HBSS, pH 7.4, 50 μL/well) before 1 × 104 cells were seeded in 100 µL DMEM per well and left to adhere overnight. After treatment with test compounds for 24 h (100 μM in 50 μL HBSS), fixation for 10 min (4% PFA/PBS, 50 μL/well) and permeabilization for 10 min (0.5% Triton X-100/PBS, 50 μL/well), the cells were stained with DAPI for 2 min (1:10,000 in 0.1% Tween-20/PBS, 15 μL/well). After washing three times for 5 min (PBST, 50 μL/well), cells were stored in PBS (50 μL) for high content imaging using an IN Cell 2200 analyser (10 × magnification, GE Healthcare, Rydalmere, NSW, Australia). Morphological changes (area and intensity) were automatically quantified for each individual nucleus. Images acquired from 4 wells with 2 images each were automatically analysed using IN Carta image analysis software (GE Healthcare, Rydalmere, NSW, Australia). Nuclear intensity was standardized on the average intensity of non-treated control nuclei and expressed as fold-change. Data represented the mean ± SEM of quadruplicates and ~1000 cells were analysed per treatment.
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4

Evaluating Cell Membrane Integrity

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To assess cell membrane integrity, as a marker of cell death, PI fluorescent staining was performed using PI dye. To stain MB spheres, 100 μL of media was removed from each well and PI at a concentration of 40 μg/mL in PBS (100 μL) was added to the treated spheres and analysed under a fluorescent microscope (IN Cell Analyzer 2000, GE Healthcare, IL, USA). ImageJ 1.49q software (NIH, Bethesda, MD, USA, website: https://imagej.nih.gov/ij/) was used to analyse PI incorporation by measuring the signal intensity of the sphere from the images. For live/dead dual staining, treated spheres were dissociated into single-cell suspension using Accutase solution (A6964, Sigma-Aldrich). Subsequently, cells were washed with PBS and stained with 3 μM calcein-AM (Thermo Fisher Scientific) and propidium iodide (5 μM) followed by incubation for 30 min. One hundred microlitres of cell suspension was transferred to black plates and imaged with fluorescent microscope (IN Cell Analyzer 2000) using excitation of 475 nm/emission of 511 nm for calcein-AM and excitation of 542 nm/emission of 620 nm for PI. Generated images were automatically analysed using IN Carta Image Analysis Software (GE Healthcare).
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5

Quantifying DNA Damage via γ-H2AX

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To assess DNA damage, 5 × 103 cells were seeded in 100 µL serum-free DMEM per well in 384-well plates (781091, µClear, Greiner, Ryde, NSW, Australia) pre-coated with rat tail collagen as described above and left to adhere overnight. The cells were treated with test compounds for 4 h (0–40 μM in 100 μL HBSS/well) while menadione was used as a positive control [32 (link)]. After fixation (50 μL) and permeabilization (50 μL) as described above, unspecific antibody binding was blocked for 1 h (5% FBS + 5% BSA in PBS, 50 μL/well) before the samples were exposed to mouse monoclonal anti-phospho-Histone H2AX (Ser139) antibody overnight (1:1000 in blocking buffer, 15 μL/well). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody for 1 h (1:10,000 in PBST, 15 μL/well), nuclei were counterstained using DAPI and stored for imaging and analysis as described above. The average numbers of γ-H2AX-positive cells were automatically quantified for all acquired images using IN Carta image analysis software (GE Healthcare, Rydalmere, NSW, Australia). Results were standardized on the non-treated control and expressed as fold-change. Data represented the mean ± SEM of quadruplicate images from 3 independent assays. At least 1000 cells were analysed per treatment.
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6

Quantifying Cellular Hsp70 Levels

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To measure cellular Hsp70 protein levels, cells were seeded, treated, fixed, and permeabilized as described under 4.6. Celastrol [83 (link)] and shikonin [84 (link)] were used as positive control compounds. After blocking, cells were exposed to rabbit monoclonal anti-Hsp70 antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-rabbit Alexa Fluor 594 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). Average cellular Hsp70 intensity was automatically quantified for each acquired image. Data were standardized on the non-treated control (100%) and expressed as mean ± SD of at least quadruplicates from one assay. At least 1 × 103 cells were analyzed for each treatment.
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7

Mitochondrial Membrane Potential Assay

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Detection of altered mitochondrial membrane potential was performed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzim-idazolylcarbocyanine iodide (JC-1) Mitochondrial Membrane Potential Assay Kit (ab113850, Abcam) according to the manufacturer’s instructions. Briefly, MB cells were washed twice and incubated with the assay solution containing JC-1 dye (10 μM) for 30 min. Cells were then washed, suspended in assay buffer (supplemented with 5% FBS) and seeded at 6 × 104 in 50 μL in a black 96-well plate (655,090, Greiner, Germany). Fifty microlitres of 2 × concentration of treatments, including different concentrations of mibefradil, NNC and carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) as a positive control, or no treatment control, was added to cells followed by incubation at 37 °C for 6 h. Cells were imaged using IN Cell Analyzer 2000 (excitation wavelength used was 475 nm and emission wavelengths were 511 nm and 587 nm for the monomer and the aggregates of JC-1 molecules, respectively). Images were analysed using IN Carta Image Analysis Software (GE Healthcare).
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8

Genotoxicity Evaluation of Test Compounds

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To assess if the test compounds induce some level of genotoxicity, cells were seeded as previously described and treated with 10 μM test compounds. Since γ-H2AX signal can decrease over time due to DNA repair, this assay captured SCQ-induced DNA damage after a short treatment period of 4 h. Celastrol was used as a positive control [87 (link)]. After fixation, permeabilization and blocking, cells were exposed to mouse monoclonal anti-phospho-Histone H2AX antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). The number of γ-H2AX-positive cells was automatically quantified for all acquired images. Percentage γ-H2AX-positive cells was expressed as mean ± SD of at least quadruplicates from one assay. At least 500 cells were analyzed for each treatment.
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9

Mitochondrial Membrane Potential Analysis

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Detection of altered mitochondrial membrane potential was performed using 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzim-idazolylcarbocyanine iodide (JC-1) Mitochondrial Membrane Potential Assay Kit (ab113850, Abcam) according to the manufacturer's instructions. Brie y, MB cells were washed twice and incubated with the assay solution containing JC-1 dye (10 μM) for 30 min. Cells were then washed, suspended in assay buffer (supplemented with 5% FBS) and seeded at 6 × 10 4 in 50 μL in a black 96-well plate. 50 µl of 2× concentration of treatments, including different concentrations of mibefradil, NNC, Carbonyl cyanide 4-tri uoromethoxy phenylhydrazone (FCCP) as a positive control, or no treatment control, were added to cells followed by incubation at 37 °C for 6 h. Cells were imaged using IN Cell Analyzer 2000 (excitation wavelength used was 475 nm and emission wavelengths was 511 nm and 587 nm for the monomer and the aggregates of JC-1 molecules respectively). Images were analysed using IN Carta Image Analysis Software (GE Healthcare).
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10

Assessing Cell Membrane Integrity by PI Staining

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To assess cell membrane integrity, as a marker of cell death, PI uorescent staining was performed using PI dye. PI is impermeable to the intact plasma membrane and therefore will only bind to DNA of cells with compromised cell membrane. To stain MB spheres, 100 μL of media were removed from each well and PI at a concentration of 40 μg/mL in PBS (100 μL) was added to the treated spheres and analysed under uorescent microscope (IN Cell Analyzer 2000, GE Healthcare, IL, USA). ImageJ 1.49q software (NIH, Bethesda, MD, USA, website: https://imagej.nih.gov/ij/) was used to analyse PI incorporation by measuring the signal intensity of the sphere from the images. For live/dead dual staining, treated spheres were dissociated into single-cell suspension using Accutase solution (A6964, Sigma-Aldrich).
Subsequently, cells were washed with PBS and stained with 3 μM calcein-AM (Thermo Fisher Scienti c) and propidium iodide (5 μM) followed by incubation for 30 min. 100 µl of cell suspension was transferred to black plates and imaged with uorescent microscope (IN Cell Analyzer 2000) using excitation of 475 nm /emission of 511 nm for calcein-AM and excitation of 542 nm /emission of 620 nm for PI. Generated images were automatically analysed using IN Carta Image Analysis Software (GE Healthcare).
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