CCK-8 was from American Peptide; fluorescent dyes were from Molecular Probes; Boc-Gln-Ala-Arg-MCA was from the Peptide Institute (Osaka, Japan); protease inhibitors were from Roche GmbH (Mannheim, Germany); IL6 quantikine enzyme-linked immunosorbent assay kit was from R&D Systems; and other reagents were from Sigma (Dorset, United Kingdom). 2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide (GSK-7975A) and pro-drug GSK-6288B were a gift from GlaxoSmithKline. CM_128 was a gift from CalciMedica. ALZET osmotic mini-pumps (2001D) were from Charles River UK, Ltd.
Fluorescent dyes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescent dyes are chemical compounds that absorb and emit light at specific wavelengths. They are used in a variety of laboratory applications, including cell staining, flow cytometry, and fluorescence microscopy, to label and visualize biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin coated 35 mm glass bottom dishes, by Eppendorf (2 mentions)
Application suite integrated software las x, by Leica (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Boc gln ala arg mca, by Peptide Institute (1 mentions)
Tcs sp8 confocal laser scanning microscopy system, by Leica (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Citrate buffer, by Merck Group (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Power block, by BioGenex (1 mentions)
Loricrin, by Fortrea (1 mentions)
Td 20 20 luminometer, by Turner Designs (1 mentions)
Ds ri2 microscope camera, by Nikon (1 mentions)
Ni e microscope c2, by Nikon (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
1 2 dioleoylphosphatidylglycerol dopg, by Avanti Polar Lipids (1 mentions)
Flow cytometry, by BD (1 mentions)
Tra 1 81, by BD (1 mentions)
Tra 1 60, by BD (1 mentions)
Type it microsatellite pcr kit, by Qiagen (1 mentions)
25 protocols using fluorescent dyes
1
Measurement of Interleukin-6 using CCK-8 Assay
2
Isolation of Murine Cardiomyocytes
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Chemicals were purchased from Sigma‐Aldrich unless otherwise stated, while fluorescent dyes were purchased from Molecular Probes (Life Technologies).
After cervical dislocation, the heart was excised into ice‐cold Tyrode solution (in mM: 120 NaCl, 20 HEPES, 5.40 KCl, 0.52 NaH2PO4, 3.50 MgCl2, 20 taurine, 10 creatine, 11 glucose [anhydrous]) containing heparin (0.2 mL of 1000 IU/mL) and transferred to retrograde Langendorff perfusion with Tyrode solution containing bovine serum albumin (BSA; 0.1%), type‐2 collagenase (250 IU/mL; Worthington) at 37°C, pH 7.4, for 15 min. The heart was then taken down and LV cardiomyocytes were isolated by dicing, agitating, and filtering in a modified ethylene glycol tetraacetic acid (EGTA)‐containing Krebs solution (in mM: 70 KOH, 40 KCl, 50l ‐glutamic acid, 20 taurine, 20 KH2PO4, 3 MgCl2, 10 glucose [anhydrous], 10 HEPES, 0.5 EGTA) with BSA (1%). After centrifugation at 300 rpm for 2 min and resuspension and incubation in Krebs solution without BSA at 37°C, pH 7.3, for 30 min, cardiomyocytes were stepwise re‐introduced to Ca2+ to reach a final suspension of Krebs solution with 1.8 mM CaCl2.
After cervical dislocation, the heart was excised into ice‐cold Tyrode solution (in mM: 120 NaCl, 20 HEPES, 5.40 KCl, 0.52 NaH2PO4, 3.50 MgCl2, 20 taurine, 10 creatine, 11 glucose [anhydrous]) containing heparin (0.2 mL of 1000 IU/mL) and transferred to retrograde Langendorff perfusion with Tyrode solution containing bovine serum albumin (BSA; 0.1%), type‐2 collagenase (250 IU/mL; Worthington) at 37°C, pH 7.4, for 15 min. The heart was then taken down and LV cardiomyocytes were isolated by dicing, agitating, and filtering in a modified ethylene glycol tetraacetic acid (EGTA)‐containing Krebs solution (in mM: 70 KOH, 40 KCl, 50
3
Monitoring Mitochondrial Function during Differentiation
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Cells cultured at different stage of differentiation (0–7–14–21 days) on fibronectin-coated 35-mm glass-bottom dishes (Eppendorf, Hamburg, Germany) were incubated for 20 min at 37 °C with either 2 μM TMRE (Tetramethylrhodamine ethyl ester perchlorate) to monitor mitochondrial membrane potential (mtΔΨ) or with 10 μM DCF-DA (2,7-dichlorofluorescin diacetate) or 5 μM MitoSox to evaluate intracellular peroxide and mitochondrial O2•− respectively. In particular, MitoSox is a probe chemically engineered to target the mitochondrial matrix by adding a liposoluble cation (TPP+) moiety to hydroethidine which, once oxidized, enhances fluorescence following intercalation in double stranded DNA. Conversely DCF, derived from DCF-DA de-acetylation by cellular esterases, is a redox-sensitive probe, trapped within the cell in the cytosol as well as into mitochondria. Fluorescent dyes were purchased from Molecular Probes (Eugene, OR, USA). Stained cells were washed with PBS and examined by a Leica TCS SP8 confocal laser scanning microscopy system as described elsewhere [35 (link)] (images collected using a 60X objective [1.4 NA]). Acquisition, storage, and data analysis were performed using the Leica Application Suite integrated software (LAS-X, Leica Microsystems, Wetzlar, Germany) and further analyzed by the ImageJ software (Wayne Rasband, NIH, USA, http://imagej.nih.gov/ij ).
4
Immunostaining of Calponin in Frozen Tissue
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The chemicals that were used in the assay were obtained from Sigma-Aldrich Co. The fluorescent dyes were purchased from Molecular Probes Inc. (Eugene, OR, USA). The tissue samples on day 28 (n=16) were embedded in optimal cutting temperature compound before they underwent frozen sectioning on a microtome-cryostat. To perform immunostaining, frozen sections were washed in PBS and blocked with 2% bovine serum albumin for 30 minutes at room temperature. The sections were then incubated for 1 hour at room temperature with primary antibodies against calponin and diluted in blocking solution. Nuclei were visualized by diamidine-2-phenylindole (DAPI) staining. The experiments were performed in triplicate.
5
Immunostaining Protocol for Collagen I
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The chemicals used in the assay were obtained from Sigma-Aldrich Corp (St Louis, MO, USA). Fluorescent dyes were purchased from Molecular Probes (Eugene, OR, USA). The tissue samples were embedded in optimal cutting temperature compound prior to being frozen sectioned using a microtome-cryostat. For immunostaining, frozen sections were washed in PBS and blocked with 2% Bovine Serum Albumin for 30 minutes, at room temperature. Sections were then incubated for 1 hour, at room temperature, with primary antibodies against type I collagen, diluted in blocking solution. Nuclei were visualized through DAPI-staining. The experiments were conducted in triplicate.
6
Intracellular ROS Detection
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Adherent cells were rinsed twice with serum-free medium and probed with fluorescent dyes (all from Molecular Probes) prepared in serum-free medium. To detect intracellular ROS, cells were stained with 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 20 min at 37°C, then treated with radiation at indicated dose. ROS levels were assessed by fluorescence microscopy.
7
Fluorescent Probes for Mitochondrial and ROS Dynamics
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All chemicals were obtained from Sigma (St. Louis, Mo., USA), and fluorescent dyes were purchased from Molecular Probes Inc. (Eugene, Oregon., USA). TMRM was used to measure mitochondrial membrane potential (100 nM) and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF) was used as a ROS probe (500 nM). Fluorescent probes were loaded at room temperature for 20–30 min. After loading, cells were rinsed three times in PBS. Cells loaded with dyes in the ester form (i.e. DCF) required an additional 30 min before use. Dye-loaded cells were mounted in a cell chamber for microscopic observation. Confocal fluorescence images were obtained using a Leica confocal microscope (model TCSNT) equipped with an Ar/Kr laser and filters specific for DCF (excitation wavelength: 450–490 nm; emission wavelength: 515–565 nm) and TMRM (excitation wavelength: 540–550 nm; emission wavelength: LP 590–600 nm). All experiments were performed at room temperature.
8
Mitochondrial Membrane Potential and ROS Evaluation
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Cells cultured at low density on fibronectin-coated 35-mm glass-bottom dishes (Eppendorf, Hamburg, Germany) were incubated for 20 min at 37 °C with (a) 2 μM TMRE (Tetramethylrhodamine ethyl ester perchlorate) or 0.5 μM MitoTracker Red to monitor mtΔΨ, (b) 10 μM DCF (dichlorofluorescein) to evaluate ROS production. Fluorescent dyes were purchased from Molecular Probes (Eugene, OR, USA). Stained cells were washed with PBS and examined using a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Acquisition, storage, and data analysis were performed using the Leica Application Suite integrated software (LAS-X, Leica Microsystems, Wetzlar, Germany) and the ImageJ software (Available online: https://imagej.nih.gov/ij/download.html (accessed on 10 April 2020)).
9
Comprehensive Reagent Procurement Protocol
10
Immunofluorescence Analysis of Mandible Sections
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Paraffin-embedded mandible sections were dehydrated, rehydrated, and then pretreated in 10 mmol/L citrate buffer (pH 6.0, Sigma-Aldrich) for 20 min using a microwave for antigen retrieval. The specimens were blocked by Power Block (BioGenex) and incubated with primary antibodies against Med1 (Santa Cruz, TRAP220), Krt71 (Progen), and loricrin (Covance), Sox2 (Epitomics), Alpl (R&D system), Notch1 (Cell Signaling), and c-Notch1 (Cell Signaling). They were subsequently incubated with species specific secondary antibodies conjugated with fluorescent dyes (Invitrogen Molecular Probes), including Alexa 594 (red) and Alexa 488 (green). They were then counterstained with DAPI. The images were taken with a confocal microscope (LSM510, Carl Zeiss). Pictures were taken under magnification of 200.
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