Pf 01247324

Manufactured by Merck Group

Sourced in Germany

PF-01247324 is a laboratory equipment product manufactured by Merck Group. It is designed for use in various research and analytical applications. The core function of this product is to facilitate accurate and precise measurements and data collection.

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Lab products found in correlation

Isoproterenol, by Merck Group (2 mentions) D8418, by Merck Group (1 mentions) Potassium chloride (kcl), by ITW Reagents (1 mentions) Cacl2, by Avantor (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Thapsigargin, by Merck Group (1 mentions) Isoproterenol iso, by Merck Group (1 mentions) Patchmaster 2, by HEKA Elektronik (1 mentions) Eclipse te2000 u, by Nikon (1 mentions) A 803467, by Merck Group (1 mentions)

6 protocols using pf 01247324

Patch-Clamp Analysis of iPSC-Derived Cardiomyocytes

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The patch-clamp experiments was performed as previously described [19 (link),22 (link)]. Briefly, 35,000 atrial iPSC-CMs were plated on glass-bottom Fluoro Dishes and incubated with either isoproterenol (50 nmol/L, Sigma-Aldrich, Taufkirchen, Germany)) or isoproterenol + PF01247324 (1 µmol/L, Sigma-Aldrich, Taufkirchen, Germany)) for 15 min before starting the measurements. The experiments were conducted at room temperature.
Action potential recordings were performed using the whole-cell patch-clamp technique. To elicit action potentials, square current pulses with amplitudes of 0.5–1 nA and durations of 1–5 ms were applied. The stimulation frequency was increased gradually from 0.5 to 2 Hz.
The late sodium current (I_NaL) was measured using the ruptured-patch whole-cell patch-clamp technique. The pipette used had a resistance ranging from 2 to 3 mega-ohms (MΩ). I_NaL recordings were performed exclusively in CMs where a seal with a resistance of over 1 giga-ohm (GΩ) was achieved, and the access resistance remained below 7 MΩ. After a stabilization period of 3 min, the iPSC-derived CMs were held at a holding potential of −120 mV and then depolarized to −35 mV for 1000 ms with 10 pulses and a basic cycle length of 2 s. The I_NaL was quantified as the integral current amplitude between 100 and 500 ms and was normalized to the membrane capacitance.

Hartmann N., Knierim M., Maurer W., Dybkova N., Hasenfuß G., Sossalla S, & Streckfuss-Bömeke K. (2023). Molecular and Functional Relevance of NaV1.8-Induced Atrial Arrhythmogenic Triggers in a Human SCN10A Knock-Out Stem Cell Model. International Journal of Molecular Sciences, 24(12), 10189.

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Reagent Preparation for Biochemical Assays

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KCl (131494; Panreac-AppliChem, Castellar del Vallès, Barcelona, Spain), CaCl₂ (22320.298; VWR, Llinars del Vallès, Barcelona, Spain), and NaCl (S9888; Sigma–Aldrich, Madrid, Spain) were freshly weighed and dissolved before each assay. Thapsigargin (T9033; Sigma–Aldrich), PF-01247324 (PZ0274; Sigma-Aldrich), BIII 890CL (Boehringer Ingelheim, Sant Cugat del Vallès, Barcelona, Spain) and BIII-55CL (Boehringer Ingelheim) were diluted in DMSO (dimethyl sulfoxide; D8418; Sigma–Aldrich) at a stock concentration of 10 mM and stored at –20°C. Tetrodotoxin citrate (T-550; Alomone Labs, Jerusalem, Israel) was diluted in water at a stock concentration of 1 mM and stored at –20°C.

Martínez A.L., Brea J., Domínguez E., Varela M.J., Allegue C., Cruz R., Monroy X., Merlos M., Burgueño J., Carracedo Á, & Loza M.I. (2022). Identification of Sodium Transients Through NaV1.5 Channels as Regulators of Differentiation in Immortalized Dorsal Root Ganglia Neurons. Frontiers in Cellular Neuroscience, 16, 816325.

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Selective Inhibition of NaV1.8 Currents

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For selective inhibition of Na_V1.8-induced sodium currents, a specific Na_V1.8 blocker PF-01247324 (1 µmol/L, Sigma-Aldrich, Taufkirchen, Germany)) was used. Cellular electrophysiological measurements were performed under slight beta-adrenergic stimulation (isoproterenol (Iso), 50 nmol/L, Sigma-Aldrich, Taufkirchen, Germany)) [20 (link)]. Prior to the start of experiments, the CMs were incubated for 15 min with both substances or isoproterenol alone as a control.

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Sodium Current Measurement in Mouse Cardiomyocytes

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Ruptured-patch whole-cell voltage-clamp was used to measure I_Na in mouse cardiomyocytes with microelectrodes (2–3 MΩ, room temperature). Pipettes were filled with (in mmol/L): 80 CsCl, 40 Cs-glutamate, 5 NaCl, 0.92 MgCl₂, 5 Mg-ATP, 0.3 Li-GTP, 5 HEPES, 0.03 niflumic acid (to block Ca²⁺-activated chloride current), 0.02 nifedipine (to block Ca²⁺ current), 0.004 strophanthidin (to block Na⁺/K⁺ ATPase current), 1 EGTA, and 0.36 CaCl₂ (free [Ca²⁺]_i,100 nmol/L) (pH 7.2 with CsOH at room temperature). Cardiomyocytes were maintained in the bath solution containing (in mmol/L): 5 NaCl, 135 tetramethylammonium chloride, 4 CsCl, 2 MgCl₂, 10 glucose, 10 HEPES, 0.4 CaCl₂ (pH 7.4 with KOH at room temperature). I_Na was measured only in those cardiomyocytes where a seal of more than 1 Giga-Ohm was achieved and the access resistance remained <7 MΩ. Mouse cardiomyocytes were incubated with PF-01247324 (1 µmol/L, Sigma) for 15 min before starting I_Na measurements. In all experiments, myocytes were mounted on the stage of a microscope (Nikon Eclipse TE2000-U). Liquid junction potentials were corrected with the pipette in the bath. Membrane capacitance and series resistance were compensated after patch rupture. Data were collected using Patchmaster 2.0 (HEKA Elektronik).

Bengel P., Dybkova N., Tirilomis P., Ahmad S., Hartmann N., A. Mohamed B., Krekeler M.C., Maurer W., Pabel S., Trum M., Mustroph J., Gummert J., Milting H., Wagner S., Ljubojevic-Holzer S., Toischer K., Maier L.S., Hasenfuss G., Streckfuss-Bömeke K, & Sossalla S. (2021). Detrimental proarrhythmogenic interaction of Ca2+/calmodulin-dependent protein kinase II and NaV1.8 in heart failure. Nature Communications, 12, 6586.

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Measurement of Persistent Sodium Current

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Ruptured whole-cell patch-clamp experiments were applied to establish the current–voltage relationship of I_NaL. The pipette resistance ranged between 1.5 and 3.5 MΩ when filled with the pipette solution containing (in mmol/L): 95 CsCl, 40 Cs-glutamate, 10 NaCl, 0.92 MgCl₂, 5 Mg- ATP, 0.3 Li-GTP, 5 HEPES, 0.03 niflumic acid (to block Ca²⁺-activated chloride current), 0.02 nifedipine (to block Ca²⁺ current), 0.004 strophanthidin (to block Na⁺/K⁺ ATPase current), 1 EGTA, and 0.36 CaCl₂ (free [Ca²⁺]_i,100 nmol/L) (pH 7.2 with CsOH at room temperature). The bath solution contained (in mmol/L): 135 NaCl, 5 tetramethylammonium chloride, 4 CsCl, 2 MgCl₂, 10 glucose, 10 HEPES (pH 7.4 with CsOH at room temperature). In some experiments, cardiomyocytes were incubated with PF-01247324 (1 μmol/L, Sigma) for 15 min before initiation of I_NaL measurements. After patch rupture cardiomyocytes were allowed to stabilize for at least 3 min. Currents were elicited using a voltage step protocol with 10 mV increments ranging from −120 to +30 mV (step duration: 1 s, holding potential: −120 mV). I_NaL was determined as the mean current density between 180 to 190 ms of each depolarization step. PF-sensitive I_NaL was calculated as the difference between the mean I_NaL densities of the vehicle and PF-treated cardiomyocytes at each membrane potential.

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Selective Blockade of Nav1.8 in Cardiomyocytes

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For selectively blocking Na_V1.8, isolated cardiomyocytes were treated with either A-803467 (30 nmol/L, Sigma) or PF-01247324 (1 µmol/L, Sigma). Cells were incubated for 15 min before measurements were started. Isoproterenol (30 nmol/L, Sigma) was used for slight beta-adrenergic stimulation in all groups [10 (link)]. Moreover, we used tetrodotoxin (2 µmol/l) to inhibit I_NaL.

Pabel S., Ahmad S., Tirilomis P., Stehle T., Mustroph J., Knierim M., Dybkova N., Bengel P., Holzamer A., Hilker M., Streckfuss-Bömeke K., Hasenfuss G., Maier L.S, & Sossalla S. (2020). Inhibition of NaV1.8 prevents atrial arrhythmogenesis in human and mice. Basic Research in Cardiology, 115(2), 20.

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