After IRI model and Apelin treatment, the hearts were rinsed off the blood, and subsequently immobilized for 48 h. The heart samples were dehydrated using different concentrations of ethanol and embedded in paraffin. The paraffin was cut into slices (5 μm) and stained with HE. IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, USA) was used to image the staining slices.
Incucyte s3 zoom cell imaging system
Manufactured by Sartorius
Sourced in United States
The IncuCyte™ S3 ZOOM is a cell imaging system that enables automated, real-time monitoring and analysis of live cells in their native environment. It captures high-quality images of cells over time, providing quantitative data on cell growth, morphology, and behavior.
Automatically generated - may contain errors
9 protocols using incucyte s3 zoom cell imaging system
1
Paraffin Embedding and Staining
2
Measurement of Intracellular Calcium in hiPSC-CMs
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Fluo-3 AM (Thermo Fisher, F1242), a calcium fluorescence indicator, was used to measure the intracellular calcium levels of hiPSC-CMs [25 (link), 26 (link)]. Following the different group treatments, hiPSC-CMs were incubated in a medium containing Fluo-3 AM (5 μM) in the dark for at least 30 min. After 3 washes with PBS, hiPSC-CMs were placed in a fresh medium for 20 min to ensure the generation of calcium fluorescence. To evaluate intracellular calcium levels, images were captured using an IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI).
3
Evaluating ROS in hiPSC-CMs
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A 96-well plate was used to seed hiPSC-CMs at a concentration of 5 × 104 cells per well. The cultured cells were treated with different concentrations of compounds and were incubated for another 48 h. Cells were incubated in a maintaining medium containing 10 μM DCFH-DA (Beyotime Biotechnology, Shanghai, China) for 30 min at 37 °C in the dark [18 (link)]. The measurement of ROS was performed with IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI). TissueQuest 6.0 was used for the quantitative analysis of ROS formation. Data were presented as the mean ± SEM, n ≥ 3.
4
Histopathological Analysis of Heart Tissue
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The heart tissue was fixed with 4% paraformaldehyde and embedded in paraffin. Subsequently, 5 μm sections were prepared and stained with hematoxylin and eosin (H&E). Then the pathological changes of heart tissue were detected using IncuCyte™ S3 ZOOM Cell Imaging System (Essen BioScience, Ann Arbor, MI, USA).
The immunohistochemical staining of NLRP3, ASC, and caspase-1 tissue sections was carried out according to the kit instructions. Briefly, the tissue sections were dewaxed and hydrated. Their antigen was retrieved, and the endogenous peroxidase was blocked. The sections were treated with goat serum and then incubated with NLRP3, ASC, and caspase-1 antibodies overnight. The slides were stained using the DAB kit, restained with hematoxylin, and observed using Aperio S2 Leica Biosystem microscope (Leica, Wetzlar, Germany). Quantification of all data was performed with ImageJ software.
The immunohistochemical staining of NLRP3, ASC, and caspase-1 tissue sections was carried out according to the kit instructions. Briefly, the tissue sections were dewaxed and hydrated. Their antigen was retrieved, and the endogenous peroxidase was blocked. The sections were treated with goat serum and then incubated with NLRP3, ASC, and caspase-1 antibodies overnight. The slides were stained using the DAB kit, restained with hematoxylin, and observed using Aperio S2 Leica Biosystem microscope (Leica, Wetzlar, Germany). Quantification of all data was performed with ImageJ software.
5
hESC-CM Calcium Dynamics Monitoring
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The hESC-CMs in this experiment can express the GCaMP reporter gene by gene editing, and green fluorescence marker can be observed by a fluorescence microscope. The fluorescence intensity is proportional to Ca2+ concentration. The CMs were seeded into 96-well plates at 5000/well and were administered the same as above [19 (link)]. The fluorescence intensities of hESC-CMs were monitored using an IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI). Data were presented as the mean ± SEM, n ≥ 3.
6
Measuring Intracellular ROS in hiPSC-CMs
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The experimental protocol of hiPSC-CM culture was the same as above. The level of intracellular ROS in hiPSC-CMs was detected using the ROS assay kit (Beyotime, Shanghai, China). Following different treatments, DCFH-DA (10 μM) probe was added to the cells for 30 min at 37 °C in the dark. Cells were washed three times to remove probes that did not enter [18] (link). Intracellular ROS level was reflected by the fluorescence intensity measured using the IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI).
7
Measuring Intracellular Calcium Dynamics
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The experimental protocol of hiPSC-CM culture was the same as above. Fluo-3 AM was used as fluorescent indicator to measure intracellular calcium levels. The cardiomyocytes were cultured to a confluence of ~ 80% and divided into groups. Then, cardiomyocytes were incubated in medium containing 5 μM Fluo-3AM at 37 °C in the dark for 40 min. After adding fresh medium, the cells were incubated for 20 min at 37 °C in the dark to generate calcium fluorescence [19] (link). Intracellular calcium levels were determined by the fluorescence images taken using IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI).
8
Quantifying Apoptosis in NPCMs via TUNEL Assay
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Apoptosis of NPCMs was determined using TUNEL assay as previously reported [27 (link), 28 (link)]. Following treatments, 0.1% Triton X-100 was used to increase membrane permeability and TUNEL reaction solution was added to the cells for 1 h at 37°C. After washing three times with PBS, images were taken using an IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI).
9
Mitochondrial Membrane Potential Analysis
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The mitochondrial membrane potential in NPCMs was detected using the JC-1 dye (Thermo Fisher, 65-0851-38) [29 (link)]. JC-1 (1 : 1000) was added to NPCMs after treatments and incubated for 30 min at 37°C. Then, cells were washed three times with PBS. Images were collected using an IncuCyte™ S3 ZOOM cell imaging system (Essen BioScience, Ann Arbor, MI).
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