Rsv was dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted with sterile water to prepare a 50% DMSO solution at 60 mM stock concentration. CF or MF medium with 10% serum was replaced with serum free media 3 h before the Rsv treatment. Cells were maintained in serum free medium until the end of the experiment. CF, MF and cardiomyocytes were treated for 24 h with a range of Rsv doses (5, 10, 30 and 60 µM) or vehicle control. Live dead cell assay was used to determine the effect of resveratrol on CF, MF and cardiomyocytes. Briefly, 24 h after treatment with Rsv, CM, MF and cardiomyocytes were incubated with calcein AM (Invitrogen, ON, Canada) and ethidium homodimer 1 (Biotium, CA, USA) for 20 min. ethidium homodimer 1 is highly positively charged and hence non-permeable in live cells. It enters cells with damaged membrane and emits red fluorescence when bound to DNA. calcein AM is a cell permeable dye that gets converted to its fluorescent form after hydrolysis by intracellular esterases and is used to detect viable cells. Red and green cells were imaged using EVOSfl fluorescent microscope (AMG, ThermoFisher Scientific, MA, USA) at a 10× magnification. The images were analyzed using ImageJ software. All analyses were done in a blinded manner.
Ethidium homodimer 1
Manufactured by Biotium
Sourced in Canada
Ethidium homodimer-1 is a fluorescent dye used for nucleic acid staining in various applications, such as gel electrophoresis and fluorescence microscopy. It binds to DNA and RNA, emitting a red-orange fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Biotium (6 mentions)
Hoechst 33342, by Biotium (5 mentions)
Mitosox, by Thermo Fisher Scientific (5 mentions)
Mitoview green, by Biotium (3 mentions)
Rhod 2 am, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Retiga srv fast 1394 camera, by National Instruments (2 mentions)
Axiocam 702 mono camera, by Zeiss (2 mentions)
Calibri 7 led light source, by Zeiss (2 mentions)
Evos fl fluorescent microscope, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258 dye, by Polysciences (1 mentions)
Dmi6000, by Leica (1 mentions)
Hoechst 33342, by Enzo Life Sciences (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Tetramethylrhodamine methyl ester tmrm, by Biotium (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using ethidium homodimer 1
1
Resveratrol Cytotoxicity Evaluation
2
Multiparametric Imaging of Cellular Stress Responses
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Hoechst 33342, TMRM, calcein-AM, and Ethidium Homodimer-1 were all purchased from Biotium and applied using manufacturer’s protocol31 (link),57 (link). MitoSOX was purchased from Life Technologies and was applied to cells for a 10-min incubation to assess mitochondrial superoxide31 (link). Calcein-Cobalt Chloride (CoCl2) staining was used to assess mitochondrial permeability transition, which is achieved through quenching cytosolic calcein-AM signal with 5 μM CoCl2 described previously31 (link). Immunofluorescence with Anti-MF-20 (DSHB # AB_2147781) was used with fluorescent secondary antibody conjugated to Alexa Fluor 555 (Themo # A-31570) to assess cardiac hypertrophy in fixed and permeabilized PVNM cells. All imaging experiments were done on an Olympus IX70 inverted microscope with QImaging Retiga SRV Fast 1394 camera using NIS Elements AR 3.0 software. Quantification, scale bars, and processing including background subtraction, was performed on Fiji (ImageJ) software.
3
Cell Viability Analysis in NP Tissue
4
Multimodal Imaging of Calcium Signaling
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TMRM, Calcein-AM, ethidium homodimer-1, MitoView Green, and Hoechst 33342 were purchased from Biotium. MitoSox was purchased from Life Technologies. MPTP imaging was performed by quenching the cytosolic Calcein-AM signal with 5 µM cobalt chloride during the incubation period. All imaging, including ER and mitochondrial calcium imaging, was done on an Olympus IX70 inverted microscope with QImaging Retiga SRV Fast 1394 camera using NIS Elements AR 3.0 software. Quantification, scale bars, and processing including background subtraction, was done on ImageJ software. ER-LAR-GECO and mito-carmine imaging was performed 48-hours following transfection in H9c2 cells and 18-hours after FSK-I stimulation (additional details are included in figure legends). Calcium imaging in ventricular myocytes was done 48-hours after viral transduction. ATeam 1.03 was imaged with a CFP and FRET (CFP-YFP) cube-set on an Olympus IX70 inverted microscope with QImaging. Images were analyzed and quantified using ImageJ software.
5
Multimodal Imaging of Mitochondrial Function
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MitoView Green, TMRM, Calcein-AM, ethidium homodimer-1, and Hoechst 33342 were purchased from Biotium. MitoSox was purchased from Life Technologies. MPTP imaging was described previously [19 (link)]. Dye based calcium imaging was done with Rhod-2AM (Invitrogen, R1245MP) as per manufacturer’s protocol (including the production of dihyrdorhod-2 AM). Immunofluorescence with HMBG1 (CST # 3935), Bnip3 [CST # 3769 and Ab-196706 (Alexa Fluor 647)], 14-3-3β [sc-25276 (Alexa Fluor 488)], and Opa1 [sc-393296 (Alexa Fluor 488)] antibodies were performed in conjunction with fluorescent secondary antibodies conjugated to Alexa Fluor 466 or 647 (Jackson), when primary antibodies were not conjugated to a fluorophore. All epifluorescent imaging experiments were done on a Zeiss Axiovert 200 inverted microscope fitted with a Calibri 7 LED Light Source (Zeiss) and Axiocam 702 mono camera (Zeiss) in combination with Zen 2.3 Pro imaging software. Confocal imaging was done on a Zeiss LSM700 Spectral Confocal Microscope in combination with Zen Black, which was also used for colocalization analysis, while FRET imaging was done using a Cytation 5 Cell Imaging Multi-Mode Reader. Quantification, scale bars, and processing including background subtraction, was done on Fiji (ImageJ) software. Quantification of mitochondrial morphology was performed as previously described [43 ].
6
Viability Assessment of HUVEC on PGSA/PEGDA
7
Hydrogel Cell Viability Assay
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To assess cell viability, the media were removed, and hydrogels were rinsed with HBSS. HBSS containing 1 µM calcein AM (Biotium, Freemont, CA, USA), 1 µM Hoechst 33342 (Enzo Life Sciences, Inc., Farmingdale, NY, USA), and 3 µM ethidium homodimer-1 (Biotium) was added to the hydrogels at a volume of 250 µL per well. After 1 h of incubation, Z-series images were obtained using a Nikon A1-R confocal microscope with 20× objective, resonance scanning, 2× averaging to reduce noise, and a Z-step size of less than 1 µm.
8
Multimodal Cell Imaging Protocol
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MitoView Green, TMRM, Calcein-AM, ethidium homodimer-1, and Hoechst 33342 were purchased from Biotium. MitoSox was purchased from Life Technologies. MPTP imaging was described previously 24 . Dye based calcium imaging was done with Rhod-2AM (Invitrogen, R1245MP) as per manufacturer's protocol (including the production of dihyrdorhod-
9
Fluorescent Imaging of Cellular Stress Pathways
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H9c2 or PVNCs were cultured and treated with DOX for 24 hours, after which they were incubated with the appropriate dye for 30 minutes, washed with PBS three times, and then imaged in culture media. All imaging was done using a Zeiss Axiovert 200 inverted microscope equipped with a Calibri 7 LED Light Source (Zeiss) and Axiocam 702 mono camera (Zeiss). Calcein-AM, ethidium homodimer-1, tetramethylrhodamine methyl ester (TMRM), and Hoechst 33342 dyes were purchased from Biotium. MitoSOX was purchased from Life technologies while LysoTrackerRed DND-99, Rhod-2AM, and dichlorodihydrofluorescein diacetate (H 2 -DCFDA; DCF) were purchased from Invitrogen. Mitochondrial permeability transition pore imaging (mPTP) imaging was carried out with Calcein-AM and cobalt chloride using the same method previously described by our lab (28, (link)30, (link)31) (link). Mito-pHred, a plasmid-based biosensor (33) (link) and dihydro-Rhod2 (28) imaging were described previously.
10
Live/Dead Assays of MEC Spheroids on Compliant Hydrogels
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Live/Dead assays of MECs on compliant PA gels were performed using a Viability/Cytotoxicity Kit (Thermo Fisher). Briefly, single round MECs grown in 2D and 3D conditions were seeded for 48hr and were washed twice in PBS prior to live/dead quantification. Live cells were stained using Calcein-AM (Thermo Fisher) and dead cells were stained using Ethidium homodimer-1 (Biotium) according to manufacturer's instructions. Percent cell death measurements were calculated based on the number of dead cells/total cells. For the 2D monolayer and spheroid live/dead assay, cells were either cultured on 2D plastic surfaces (2D monolayer) or embedded within rBM and grown for 12 days to form 3D spheroids. Cells were treated in the absence or presence of recombinant, purified human or mouse TRAIL (PeproTech), Paclitaxol or doxorubicin. The percent of live/dead cells in 2D monolayers and organoids was determined by quantifying percent active caspase-3 cells by indirect immunofluorescence, normalizing dead cells to total cells enumerated from nuclei counterstained with Hoechst 33342.
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