Single fibers were prepared from FDB muscles of YS and WT mice according to a modified collagenase-dissociation method previously described (Defranchi et al., 2005 (link)). Briefly, FDB muscles were dissected and incubated at 37°C for 1–2 h in 0.2% collagenase in Tyrode’s solution (Sigma–Aldrich, Milano, Italy) containing 10% fetal bovine serum (Sigma–Aldrich, Milano, Italy). After digestion, collagenase was removed by 3 min wash with Tyrode’s solution, then muscles were incubated for 15 min in Tyrode’s solution containing 10% fetal bovine serum to completely block the collagenase activity, and finally washed again for 3 min in Tyrode’s solution. Fibers were dissociated using Pasteur pipettes of decreasing diameter, plated on laminin-coated glass coverslips positioned at the center of 35-mm dishes, and incubated overnight at 37°C in Tyrode’s solution supplemented with 10% heat-inactivated FBS and 1% antibiotic–antimycotic solution (10,000 units penicillin, 10 mg streptomycin, and 25 μg amphotericin B) (Sigma–Aldrich, Milano, Italy).
Tyrode s solution
Manufactured by Merck Group
Sourced in United States, Italy
Tyrode's solution is a physiological salt solution commonly used in cell culture and laboratory research. It is a balanced salt solution that maintains the appropriate pH, osmolarity, and ionic composition to support the survival and function of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (7 mentions)
Pluronic acid, by Thermo Fisher Scientific (4 mentions)
Fluo 3 am, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Ix83xdc, by Olympus (3 mentions)
Egg yolk lecithin, by Merck Group (3 mentions)
Probenecid f 127, by Thermo Fisher Scientific (3 mentions)
Fluo 4 nw dye, by Thermo Fisher Scientific (3 mentions)
Live cell uorescence microscope, by Olympus (2 mentions)
Accutase medium, by Thermo Fisher Scientific (2 mentions)
Carboxyl chitosan, by Merck Group (2 mentions)
Rhodamine b, by Merck Group (2 mentions)
Rnasin, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Imagexpress micro xls microscope, by Molecular Devices (2 mentions)
Milrinone, by Merck Group (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
M2 medium, by Merck Group (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
83 protocols using tyrode s solution
1
Isolation of Single Muscle Fibers
2
Patch-clamp Electrophysiology Protocol
3
Functional Characterization of Electrically Stimulated Myocytes and Motoneurons
4
Derivation and Culture of Stem Cells from Blastocysts
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After the culture of semi-cloned embryos in KSOM medium for 4–5 days, each blastocyst was transferred on MEFs in serum plus LIF medium, which component was previously described [34 ]. For unhatched blastocysts, zona pellucida was removed by the treatment with the Tyrode’s solution (Merck) before the transfer. After the expansion of blastocyst outgrowth, cells were maintained on MEFs until the passage 5. After the passage 5, scESCs were maintained on gelatin-coated plates without MEFs in serum plus LIF medium. Karyotyping by flow cytometry and chromosome counting was performed for scESCs at the passage 9.
5
Single-Cell Isolation Protocol for Cleavage-Stage Embryos
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The ZP was removed from each embryo by exposure to acidified Tyrode's solution (EMD Millipore) and washed with Ca2+- and Mg2+-free phosphate buffered saline (PBS). Cleavage-stage embryos were disaggregated into single cells, polar bodies, and cellular fragments if present with Quinn's advantage Ca2+- and Mg2+-free medium with HEPES plus 10% human albumin (CooperSurgical) and 0.05% trypsin-EDTA (Thermo Fisher Scientific) as necessary. Each blastomere, polar body, and cellular fragment was washed with Ca2+- and Mg2+-free PBS and collected individually for transfer to a sterile UltraFlux PCR tube (VWR). All of the above was performed under a stereomicroscope equipped with a digital camera (Leica Microsystems), which has movie-making capabilities, to document the collection of every sample. Samples were put into tubes, flash frozen on dry ice, and stored at −80°C. Only embryos for which the disassembly process occurred effectively with no apparent loss of material were carried forward for library preparation and sequencing.
6
Pharmacological Assessment of Vasodilator Mechanisms
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All the chemicals were 99.9% pure and of research-grade quality. Acetylcholine, carbachol, doxazosin, glibenclamide, isoprenaline, loperamide hydrochloride, phenylephrine, and potassium chloride were obtained from Sigma Chemical Co. (St Louis, MO, USA), while cromakalim was procured from Tocris, Ellisville, MN, USA. Castor oil was purchased from Karachi Chemicals (Karachi, Pakistan). The chemicals used to make the physiological solutions, i.e., Krebs solution and Tyrode’s solution, were procured from Merck (Dermstadt, Germany) and BDH (Poole, England).
7
Measuring Neuronal Calcium Dynamics
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In order to identify potential of neuronal differentiation, we evaluated calcium in ux which is an indicator for neurotransmitter transmission. The intracellular calcium assessment was described in previous study (23) . The specimens were incubated with 3 µM Fluo-3 AM (Invitrogen) and 0.08% pluronic acid (Invitrogen) in DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin at 37 o C for 60 minutes. Subsequently, the specimens were washed with DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and PBS. The specimens were maintained in Tyrode's solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 30 mM glucose, and 25 mM HEPES, pH 7.4) (All from Sigma-Aldrich). The neurotransmitter releasing ability of differentiated cells was simulated with 50 mM KCI. The intensity of calcium was recorded time-lapse at excitation 506 nm for 3 minutes by the Live-cell uorescence microscope, IX83XDC (Olympus), and interpreted using the ImageJ program (NIH).
8
Evaluating Neuronal Calcium Dynamics
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In order to identify potential of neuronal differentiation, we evaluated calcium in ux which is an indicator for neurotransmitter transmission. The intracellular calcium assessment was described in previous study (23) . The specimens were incubated with 3 µM Fluo-3 AM (Invitrogen) and 0.08% pluronic acid (Invitrogen) in DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin at 37 o C for 60 minutes. Subsequently, the specimens were washed with DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and PBS. The specimens were maintained in Tyrode's solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 30 mM glucose, and 25 mM HEPES, pH 7.4) (All from Sigma-Aldrich). The action potential ability of differentiated cells was simulated with 50 mM KCI. The intensity of calcium was recorded time-lapse at excitation 506 nm for 3 minutes by the Live-cell uorescence microscope, IX83XDC (Olympus), and interpreted using the ImageJ program (NIH).
9
Contractile Response of bMTFs to Acetylcholine
10
Calcium Transients in hiPSC-Derived Cardiomyocytes
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hiPSC-CMs were purchased from Ncardia (Cor.4U cardiomyocytes, Ax-C-HC02-96) as living pre-plated cells seeded onto fibronectin-coated 96-well μClear plates (Greiner Bio-One, No. 655090) at a density (∼25,000 cells/well) suited to forming a confluent synchronously beating monolayer. Cor.4U cardiomyocytes represent a mix of 60% ventricular, 30% atrial, and 10% nodal cells according to the cell provider. Cells were cultured with Cor.4U culture medium (Ax-M-HC250) in a humidified incubator at 37°C and 5% CO2, with medium being changed once a day. On the day of the experiment, the culture medium was replaced with Tyrode's solution (Sigma, No. T2397) supplemented with 10 mM HEPES together with KCl to represent isokalemic (4.2 mM K+) conditions.
Table S1 contains the purchase information and free Cmax references for all the compounds used within this work. The preparation of drug solutions is explained in Supplemental Experimental Procedures . The calcium-sensitive fluorescence dye Cal-520 AM (Cat. No. 36,338; AAT Bioquest) was used to capture the intracellular calcium transients in hiPSC-CMs. The protocol was used as described in Kopljar et al. (2018) (link). Briefly, Cal-520 was incubated for 45 min followed by a washout and a 30-min recovery before starting the experiments.
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