Fasudil

Manufactured by LC Laboratories

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Fasudil is a laboratory product manufactured by LC Laboratories. It is a chemical compound used for research purposes.

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Fasudil hydrochloride (3 citations)

18 protocols using fasudil

Hepatic Injury Protocols: DEN, Fasudil, AT13148

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To prepare diethylnitrosamine (Sigma, N0258), 100 mL of sterile PBS was injected into the bottle containing 1 g of DEN to give a final concentration of 10 mg/mL. The procedure was performed in the fume hood. For short-term studies assessing DEN-induced hepatic injury, mice were treated with a single dose of DEN at 100 mg/kg body weight by intraperitoneal injection.
To prepare fasudil, 1 g of fasudil (LC Laboratories, Woburn, MA, USA, F-4660) was dissolved in 50 mL distilled water giving a final concentration of 20 mg/mL. fasudil was then aliquoted and stored at −20 °C until required for dosing. Mice were dosed with 100 µL (2 mg) fasudil twice daily for the duration of the study.
To prepare AT13148 (Selleckchem, S7563), compound was dissolved in 89% H₂O/10% (v/v) DMSO (Sigma, D2650)/1% (v/v) Tween 20 (Sigma, P1379) at a concentration of 8 mg/mL. The dissolved drug was kept at 4 °C for a maximum of 2 weeks. Mice were dosed three times per week (Monday, Wednesday, Friday) for the duration of studies performed. Mice were dosed initially with 30 mg/kg for the first two weeks, then 40 mg/kg thereafter.

Naylor G., Julian L., Watson-Bryce S., Mullin M., Nibbs R.J, & Olson M.F. (2022). Immunogenic Death of Hepatocellular Carcinoma Cells in Mice Expressing Caspase-Resistant ROCK1 Is Not Replicated by ROCK Inhibitors. Cancers, 14(23), 5943.

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Fasudil Treatment in mdx Mice

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Animal procedures were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethical Committee of the Universitat Autònoma de Barcelona. The animals used in this study were all 7-weeks old and males. A total of 6 DBA/2J-mdx males were treated with fasudil (LC Laboratories) at 100 mg/kg/day orally for 6 weeks. Five untreated DBA/2J-mdx males were used as controls and 5
DBA/2J males were used as healthy mice.

Fernández‐Simón E., Suárez‐Calvet X., Carrasco‐Rozas A., Piñol‐Jurado P., López-Fernández S., Bech‐Serra J.J., Torre C.d.l., Luna N.d., Gallardo E., & Jordi-Díaz-Manera. (2021). RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells in DMD.

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Fasudil Treatment in Muscular Dystrophy

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Animal procedures were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethical Committee of the Universitat Autònoma de Barcelona. The animals used in this study were all 7‐week‐old males. Six dba/2J‐mdx males were treated with fasudil (LC Laboratories) that was diluted in water and administered at 100 mg/kg/day orally for 6 weeks by gavage, according to previous studies.²⁶, ²⁷, ²⁸, ²⁹, ³⁰ Five untreated dba/2J‐mdx males were used as controls and six dba/2J males were used as healthy controls.

Fernández‐Simón E., Suárez‐Calvet X., Carrasco‐Rozas A., Piñol‐Jurado P., López‐Fernández S., Pons G., Bech Serra J.J., de la Torre C., de Luna N., Gallardo E, & Díaz‐Manera J. (2022). RhoA/ROCK2 signalling is enhanced by PDGF‐AA in fibro‐adipogenic progenitor cells: implications for Duchenne muscular dystrophy. Journal of Cachexia, Sarcopenia and Muscle, 13(2), 1373-1384.

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Intravenous Fasudil Administration Protocol

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fasudil monohydrochloride (fasudil; LC Laboratories, Woburn, MA, USA) was prepared in saline (10 mg ml⁻¹ sodium chloride 0.9% PF injection; Pencol Compounding Pharmacy, Denver, CO, USA) and passed all measures for purity by HPLC, sterility, endotoxins and fungal presence (Analytical Research Laboratories, Oklahoma City, OK, USA) prior to use. Sixty milligrams of fasudil (6 ml vial) was added to a 100 ml saline bag immediately prior to administration, covered to protect it from exposure to light, and infused intravenously over 60 min (Shibuya et al., 2005 (link)). This single dose of fasudil was well‐tolerated by both young and older adults, and no adverse events were observed or reported in either age group. For the placebo control visit, administration of saline was identical to that of fasudil, with a covered 100 ml saline bag infused intravenously over 60 min. fasudil mixed in saline was indistinguishable from saline alone, and thus all investigators and subjects remained blinded during treatment administration.

Racine M.L., Terwoord J.D., Ketelhut N.B., Bachman N.P., Richards J.C., Luckasen G.J, & Dinenno F.A. (2022). Rho‐kinase inhibition improves haemodynamic responses and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older adults. The Journal of Physiology, 600(14), 3265-3285.

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Transverse Aortic Constriction Mouse Model

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TAC was conducted in 10–15-week-old adult male mice. The thymus was retracted to expose the transverse aorta. Between the right innominate and left carotid artery, an aortic constriction was achieved by tying a 6-0 suture against a 3 mm length of 27 gauge needle. After two knots, the 27 gauge needle was promptly removed, which yielded a constriction of ∼0.3 mm as the outer diameter of the 27 gauge needle. The procedure imposed a 60–80% aortic constriction on the animal. To minimize the variation of the constriction among the animals, only the mice with a ratio of right-to-left carotid artery flow velocity from 5 : 1 to 10 : 1 were used. The artery flow velocity was measured by a Pulsed Doppler (Indus Instruments, Houston, TX, USA). As a control, a sham operation without occlusion was performed on age-matched littermate mice. For the rescue experiment, mice were allowed access to drinking water containing fasudil (∼100 mg/kg/day; LC Laboratories, Woburn, MA, USA) for 3 weeks starting immediately after TAC surgery. All of the TAC-induced pressure overload stress lasted for 3 weeks.

Yue X., Yang X., Lin X., Yang T., Yi X., Dai Y., Guo J., Li T., Shi J., Wei L., Fan G.C., Chen C, & Chang J. (2014). Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure. Cell Death & Disease, 5(6), e1284-.

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Murine Hematopoietic Stem Cell Isolation

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Fasudil was purchased from LC Laboratories (Woburn, MA). Busulfan, Cremaphor were purchased from Sigma-Aldrich (St. Louis, MO). Flourochrome-labeled monoclonal antibodies to CD45.1 and CD45.2 were purchased from BD Bioscience (San Jose, CA). The murine recombinant cytokines stem cell factor (SCF), thromobopoieitn (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L) were obtained from PeproTech (Rocky Hill, NJ). X-VIVO medium was from Lonza (Basel, Swiss). Antibodies to myosin light chain 2 (MLC) were purchased from Cell Signaling Technology (Danvers, MA).

William B.M., An W., Feng D., Nadeau S., Mohapatra B.C., Storck M.A., Band V, & Band H. (2015). Fasudil, a clinically-safe ROCK Inhibitor, Decreases Disease Burden in a Cbl/Cbl-b Deficiency-Driven Murine Model of Myeloproliferative Disorders. Hematology (Amsterdam, Netherlands), 21(4), 218-224.

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Evaluating Anti-seizure Drugs in Rats

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Rats implanted with ventral hippocampal electrodes and dorsal hippocampal optodes were used in these studies. Rats were reused for several drug experiments allowing at least two days between drug treatments. Celecoxib, SC-560, ibuprofen, chelerythrine chloride, milrinone, sildenafil, SKA-31, CAY-10526, seratrodast, ozagrel, paxilline, 2-APB, levetiracetam, and topiramate were obtained from Cayman Chemicals (Ann Arbor, MI). Acetaminophen, nifedipine, bumetanide, ethosuximide, phenytoin, and valproic acid were obtained from Sigma-Aldrich. Lamotrigine was obtained from SelleckChem (Houston, TX). Fasudil was obtained from LC laboratories (Woburn, MA) and phenobarbital was obtained from Strathcona Prescription Center (Canada). Lipophilic drugs were dissolved in 100% DMSO, while hydrophilic drugs were dissolved in saline and injected 30 min prior to seizure induction. The seizure duration and severity of hypoxia (area below 10 mmHg) were compared across kindle (seizure without injection), vehicle-, and drug-treated groups using a within-subject ANOVA and follow-up t-test between vehicle- and drug-treated groups.

Farrell J.S., Gaxiola-Valdez I., Wolff M.D., David L.S., Dika H.I., Geeraert B.L., Rachel Wang X., Singh S., Spanswick S.C., Dunn J.F., Antle M.C., Federico P, & Teskey G.C. (2016). Postictal behavioural impairments are due to a severe prolonged hypoperfusion/hypoxia event that is COX-2 dependent. eLife, 5, e19352.

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Provirus Removal from iPSCs

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LUMC043iRAG18 was selected for removal of the inserted provirus. To this end the iPSC line was first adapted to single-cell passaging as follows. iPSC colonies were dissociated into single cells or small clumps of cells (1–5 cells) by a 1–3 min incubation with TrypLE select (Thermo Fisher Scientific) and replated in 50% fresh HESCM and 50% old HESCM in which the cells were cultured in the presence of 10 μM Fasudil (LC Laboratories). Irradiated CD1 MEFs were seeded on 0.1% gelatin-coated plates 1 day before passaging. Cells were passaged when they were subconfluent, i.e., iPSC colonies almost touching (either next day or after 2 days). The provirus was removed by transduction of single cells in suspension with hcAd.FLPe.F50 adenoviral vector (Gonçalves et al., 2008 (link)) at a multiplicity of infection (MOI) of 20 transduction units (TU)/mL. After 1 h at 37°C, single cells were seeded onto MEFs. Removal of the provirus was determined by PCR analysis of the genomic DNA isolated from single-cell-derived clones 11–12 days after transduction.

Themeli M., Chhatta A., Boersma H., Prins H.J., Cordes M., de Wilt E., Farahani A.S., Vandekerckhove B., van der Burg M., Hoeben R.C., Staal F.J, & Mikkers H.M. (2020). iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests. Stem Cell Reports, 14(2), 300-311.

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Screening of Protein Kinase Inhibitors for TNBC

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The fifty-five protein kinase inhibitors (PKIs) were purchased from following sources: BML-275, FR 180204, IKK16, GW 843682X, NSC 109555, NU7441, PD407824, PF 573228, SB 218078, TCS PIM-1-1, TCS PIM-1-4a, and TPCA-1 from Tocris Biosciences (Bristol, UK); indirubin-3′-monoxime and Ro-31-8220 from Calbiochem (San Diego, CA, USA); A-769662, bosutinib, chelerythrine, CP690550, fasudil, gefitinib, imatinib, nilotinib, PKC412, roscovitine, SNS-314, and tozasertib from LC Laboratories (Woburn, MA, USA); AT7867, AT9283, AZD1152, AZD1480, BI 2536, BIX 02189, CHIR-99021, CI-1040, CYC116, danusertib, enzastaurin, GDC-0879, INCB018424, JNJ-7706621, KU-55933, LY2228820, MLN8237, PD-0325901, PF-4708671, PLX-4032, PLX-4720, SB216763, SNS-032, SP600125, VX-702, Y-27632, and ZM447439 from Selleck Chemicals (Houston, TX, USA); U0126 from Promega (Madison, WI, USA); TBCA from Millipore (Burlington, MA, USA).
All TNBC cells in this study were obtained from the American Type Culture Collection (Manassas, VA, USA). The cultured cells were monitor by trypan blue cell counting as described previously [58 (link)].

Yi Y.W., You K.S., Han S., Ha I.J., Park J.S., Lee S.G, & Seong Y.S. (2022). Inhibition of IκB Kinase Is a Potential Therapeutic Strategy to Circumvent Resistance to Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer Cells. Cancers, 14(21), 5215.

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Preclinical Study of Fasudil in Mice

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Sprague–Dawley rats and wild-type C57BL/6J mice were purchased from Charles River. Nestin-GFP mice were provided by Dr Grigori Enikolopov at Cold Spring Harbor Laboratory. B6.Cg-Tg(Nes-cre)1Kln/J mice (Stock No: 003771) and B6.129X1-Gt(ROSA)26Sor^tm1(EYFP)Cos/J mice (Stock No: 006148) were purchased from Jackson Laboratory. Eight- to twelve-week-old male mice and 12-week-old male rats were used in this study. We treated the mice with Fasudil (LC Laboratories) at a dose of 30 mg kg⁻¹ dissolved in water by an oral gavage twice daily for 4 weeks. Vehicle-treated mice received water. All animals were maintained in the animal facility of the Johns Hopkins University School of Medicine. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University, Baltimore, MD, USA.

Li C., Zhen G., Chai Y., Xie L., Crane J.L., Farber E., Farber C.R., Luo X., Gao P., Cao X, & Wan M. (2016). RhoA determines lineage fate of mesenchymal stem cells by modulating CTGF–VEGF complex in extracellular matrix. Nature Communications, 7, 11455.

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