(3-aminopropyl)triethoxysilane (APTES), glutaraldehyde solution (70% in H2O), dithiothreitol (DTT), bovine serum albumin (BSA), urea, guanidine hydrochloride (GuHCl), Sodium dodecyl sulfate (SDS) (−)-Epigallocatechin gallate (EGCG), curcumin from Curcuma longa (Turmeric), fasudil hydrochloride, baicalein, Anle138b, dopamine hydrochloride, thioflavin T (ThT), L-ascorbic acid, ampicillin, isopropyl β-d-1-thiogalactopyranoside (IPTG), phenylmethylsulfonyl fluoride (PMSF), biotin, mineral oil, PBS, HEPES, and Tris buffer were purchased from Sigma-Aldrich. NeutrAvidin biotin-binding protein, Dynabeads™ M-270 Epoxy beads were purchased from Thermo Scientific. Recombinant human α-synuclein protein monomer and recombinant human α-synuclein protein aggregate (seeds) were prepared following the previous method.66 (link)
Fasudil hydrochloride
Manufactured by Merck Group
Sourced in United States
Fasudil hydrochloride is a laboratory reagent produced by Merck Group. It is a white crystalline powder that functions as a Rho-kinase inhibitor.
Automatically generated - may contain errors
Lab products found in correlation
Guanidine hydrochloride, by Merck Group (1 mentions)
Mineral oil, by Merck Group (1 mentions)
Tris buffer, by Merck Group (1 mentions)
Dynabeads m 270 epoxy beads, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Baicalein, by Merck Group (1 mentions)
U46619, by Cayman Chemical (1 mentions)
Sphingosine 1 phosphate (s1p), by Cayman Chemical (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
96 well round bottom plate, by Sarstedt (1 mentions)
0.22 μm syringe filter, by Thermo Fisher Scientific (1 mentions)
Palbociclib, by Merck Group (1 mentions)
Tamoxifen tam, by Merck Group (1 mentions)
4 protocols using fasudil hydrochloride
1
α-Synuclein Aggregation Assay Protocol
2
Preparation of Vasomotor Agents
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Sphingosine-1-phosphate, U46619, and Y-27632 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Fasudil hydrochloride, albumin, and all other drugs and chemicals used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were made by dissolving the chemicals in 0.3 N sodium hydroxide (S1P), in 5% charcoal treated albumin (S1P), DMSO (U46619), distilled water (Y-27632, fasudil), or isotonic salt solution (PE, ACh).
3
Fasudil Hydrochloride Cytotoxicity Evaluation
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After resuspending the cryovial contents in 1 mL of cell culture media, 50 μL of cell suspension was placed into each well of round bottom 96 well plates (Sarstedt). Fasudil hydrochloride (Sigma) solutions were then prepared at 2× the desired concentration in cell culture media and sterile filtered with a 0.22 μm syringe filter (Fisher Scientific). 50 μL was added to each well. The final fasudil concentration used varied from 40 μM to 1.25 μM. Cells were then incubated at 37 °C and 5% CO2 for 4 h (timepoint selected after performing optimisation experiments). The plate was subsequently centrifuged at 300g and the media was changed to regular cell culture media (see ESI† ). Cell health assessments (such as cell recovery) were performed 24 hours post-thaw. The minimum number of technical replicates per condition was 3.
4
Conditional Genetic Manipulation in Mice
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All mouse experiments were reviewed and approved by the Yale University Institutional Animal Care and Use Committee (IACUC) and the ethical committee of the CHU Sainte-Justine Research Centre. Alk5flox, Smad2flox, Smad3flox (kindly provided by M. Matzuk), Smad4flox, β-Cateninflox(ex3) and mT/mG reporter mice (Muzumdar et al., 2007 (link)) were crossed with Cdh5CreERT2 mice (kindly provided by R. Adams), R26CreERT2 (Ventura et al., 2007 (link)), or Mfsd2aCreERT2 (Pu et al., 2018 (link)) mice. To induce postnatal gene deletion, mice were injected intragastrically with 100μg of tamoxifen (TAM) (Sigma, T5648; 25 mg/ml in corn oil) for three consecutive days and analyzed as indicated. TAM-treated, Cre-negative littermates were used as controls. For lineage tracing studies using the Mfsd2aCreERT2 line the mice received 100μg of TAM 24 h prior to sacrifice. The ROCK inhibitor Fasudil-Hydrochloride (CDS021620, Sigma) was dissolved in sterile PBS and injected intraperitoneally (20ug/g). The cell-cycle inhibitors Palbociclib (PD 0332991 isethionate, PZ0199, Sigma) was dissolved in NaLactate and administered intragastrically daily (50μg/g), while Mitomycin C (AC226940020, Fisher Scientific) was dissolved in sterile H2O and administered intraperitoneally once at P7 (20ul/g).
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