Pharmacological reagents were used at final concentrations of 10 μM nimodipine (Nim; N150; Alomone Labs, Jerusalem, Israel), 10 μM dantrolene (Dan; D9175; Sigma-Aldrich), 30 μM CPA (C-750; Alomone Labs), 3 μM ω-conotoxin GVIA (ConoGVIA; C-300; Alomone Labs), 200 nM ω-agatoxin IVA (AgaIVA; STA-500; Alomone Labs), 30 μM NiCl2 (Ni2+; 22387; Sigma-Aldrich), 10 μM ionomycin (407951; Sigma-Aldrich), 10 μM rotenone (R8875; Sigma-Aldrich), and 50 mM KCl (P9333; Sigma-Aldrich). Reagent stocks (1000×) were prepared in DMSO (Nim, Dan, CPA, ionomycin, rotenone) or water (ConoGVIA, AgaIVA, Ni2+) and stored at −20°C. KCl was prepared as a 4 M HEPES-buffered stock solution.
P9333
Manufactured by Merck Group
Sourced in United States
P9333 is a laboratory equipment product manufactured by Merck Group. It serves as a core function in scientific research and analysis processes. Detailed specifications and intended use are not included in this response.
Automatically generated - may contain errors
Lab products found in correlation
S3139, by Merck Group (2 mentions)
S9390, by Merck Group (2 mentions)
M2670, by Thermo Fisher Scientific (2 mentions)
Phr1511, by Merck Group (2 mentions)
A2383, by Merck Group (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Conogvia c 300, by Alomone (1 mentions)
Rotenone, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Brij 35, by Thermo Fisher Scientific (1 mentions)
Superose 6 10 300 gl column, by GE Healthcare (1 mentions)
Vivaspin concentrator, by Sartorius (1 mentions)
Powerlab 16sp, by ADInstruments (1 mentions)
Micrometer, by Mitutoyo (1 mentions)
D desthiobiotin, by Merck Group (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
D0632, by Merck Group (1 mentions)
Streptrap column, by GE Healthcare (1 mentions)
Hepes, by Merck Group (1 mentions)
7 protocols using p9333
1
Pharmacological Modulation of Neuronal Signaling
2
Affinity Purification of APC Protein
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Cold APC purification buffer [100 mM NaPi, Sigma #S3139 and Sigma #S9390, 300 mM KCl, Sigma #P9333, 5 mM MgCl2 × 6 H2O, Sigma #M2670, 0.001% Brij35 Thermo Fisher Scientific, #28316, 2.5 mM dithiothreitol (DTT), Sigma #D0632, 2.5 mM EDTA, Sigma #EDS] supplemented with protease inhibitors (Roche, #5056489001) and deoxyribonuclease I (DNaseI) (Roche, #10104159001) was added to frozen SF21 cell pellets expressing an APC construct. The pellet was thawed in a room temperature water bath and resuspended. The lysate was clarified by centrifugation (184.000g, 45 min, 4°C), and the supernatant was applied to a 5-ml HiTrap immunoglobulin G Sepharose FF column (GE Healthcare, #28-9083-66). After washing with APC purification buffer, GST (glutathione S-transferase)–TEV protease was added to the column, and the column was left at 4°C overnight to cleave APC off its column-bound affinity and solubility tags. The next day, APC was eluted with APC purification buffer and labeled with SNAP-reactive dye (NEB #S9105S). After SNAP labeling, APC was concentrated using Vivaspin concentrators (Sartorius), ultracentrifuged (280,000g, 10 min, 4°C), and gel filtered using a Superose6 10/300 GL column (GE Healthcare). Peak fractions were pooled, and the labeling ratio was determined using NanoDrop (Thermo Fisher Scientific). The protein was aliquoted on ice and snap frozen in liquid nitrogen.
3
Bladder Dome Contractility Assay
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Longitudinal strips of the posterior wall of the bladder dome were mounted in organ baths (5 mL) containing Krebs solution and bubbled with 5% CO2 and 95% O2 (37 °C). One hook was suspended from a transducer (type 45196 A; San-ei Instruments, Tokyo, Japan), and the lower hook was fixed to a plastic support leg attached to a micrometer (Mitutoyo, Tokyo, Japan). Each strip was equilibrated unstretched for 40 minutes. A load of 2.0 g was applied to each strip by micrometer adjustment and the load was readjusted to this level 30 minutes later. Changes in the tone of the strips were measured isometrically using force transducers and the data were recorded using LabChart v7.3.8 software and a PowerLab/16sp data acquisition system (AD Instruments, Castle Hill, Australia). Contractions by the KCl (P9333; Sigma-Aldrich, St. Louis, MO, USA; 80 mM), ATP (A2383; Sigma-Aldrich; 1 mM), EFS (1, 2, 4, 8, 16, and 32 Hz), and carbachol (PHR1511; Sigma-Aldrich; 1 nM to 1 mM) were recorded. For EFS stimulation, an electrical pulse (1 millisecond pulse width and 80 V in the bath) was delivered using a stimulator (D-7806, Hugo Sachs Elektronik, Germany) for 5 seconds at increasing frequencies (1, 2, 4, 8, 16 and 32 Hz), followed by intervals of stimulation of 5 minutes. All bladder strips were normalized to weight per 1 g.
4
Purification and Labeling of APC-ARM Construct
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Cold APC purification buffer (100 mM NaPi, Sigma #S3139 and Sigma #S9390, 300 mM KCl, Sigma #P9333, 5 mM MgCl2 × 6 H2O, Sigma #M2670, 0.001% Brij35 Thermo Fisher Scientific #28316, 2.5 mM DTT, Sigma #D0632, and 2.5 mM EDTA, Sigma #EDS) supplemented with protease inhibitors (Roche, #5056489001) and DNaseI (Roche, #10104159001) was added to frozen E. coli cell pellets expressing an APC-ARM construct. The pellet was thawed and resuspended. The lysate was clarified by centrifugation (184,000g, 45 min, 4°C), and the supernatant was applied to a StrepTrap column (GE Healthcare, 28-9075-48). The column was washed with APC purification buffer, and the protein was eluted in APC purification buffer supplemented with 10 mM d-desthiobiotin (Sigma, D1411-1G). Next day, the protein was incubated with 3C protease (Speed Biosystems, YCP1208) for 1 hour at 16°C. After SNAP labeling, the protein was concentrated and ultracentrifuged as described above and gel filtered using a GE Superdex200 10/300 column. Peak fractions were pooled, and the labeling ratio was determined using NanoDrop. The protein was aliquoted on ice and snap frozen in liquid nitrogen.
5
Fabrication and Characterization of Nanopores
6
Evaluating Bladder Function in Rat Model
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One week after M-MSC injection, the experimental groups were evaluated by awake cystometry. Bladder voiding function was evaluated in unrestrained, awake-state rats in metabolic cages as previously described [9 (link), 10 (link), 12 ]. Detrusor pressure was defined as [intravesical pressure (IVP) – intra-abdominal pressure (IAP)]. The contractility of the bladder tissues was measured by the organ bath study, as previously described [12 , 19 (link)]. In brief, longitudinal strips of posterior bladder wall were normalized to weight per 1 g and then mounted in 5 mL organ baths containing Krebs solution (118 mM NaCl, 5.0 mM KCl, 2.5 mM CaCl2, 1.0 mM MgSO4, 30 mM NaHCO3, 1.0 mM KH2PO4, and 11.4 mM glucose, pH 7.4), and maintained at 37 °C with 5% CO2 and 95% O2 continuously supplied. The contractile response to 80 mM KCl (P9333; Sigma-Aldrich, St. Louis, MO, USA), EFS (electrical field stimulation, 1, 2, 4, 8, 16, and 32 Hz), 1 mM ATP (A2383; Sigma-Aldrich), or carbachol concentration (PHR1511; Sigma-Aldrich; 1 nM to 1 mM) were recorded as previously described [12 , 19 (link)]. An electrical pulse (1 millisecond pulse width and 80 V in bath) was delivered using a stimulator (D-7806; Hugo Sachs Elektronik, Germany) for 5 s at increasing frequencies (1, 2, 4, 8, 16, and 32 Hz), with 5 min intervals between electrical field stimulations.
7
Nanopore Characterization and Wetting
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