Whole decellularized lungs were mounted in a sterile bioreactor containing endothelial culture medium and perfused at 20 ml/min overnight via the pulmonary artery (Yuan et al., 2019 (link); Engler et al., 2019 (link); Calle et al., 2011 (link)). PMECs or human iPSC-ECFCs were suspended into culture medium supplemented with 30 μM Y27632 (Cayman) at a concentration of 250,000 cells/mL. Lungs were first seeded with 50 million PMECs from the pulmonary vein followed by seeding in the same manner the rest 50 million cells into the pulmonary artery. Thereafter, arterial perfusion was initiated with a peristaltic pump at a flow rate of 4 ml/min. This perfusion rate was maintained for 4 h and was ramped up 4 ml/min every 15 min until reaching 20 ml/min. During the ensuing 7 days of culture, half of the medium (endothelial culture medium +10 μM Y27632 (Cayman)) was replaced every day. For the lipopolysaccharide (LPS) study, ROCK inhibitor was removed by complete medium change on day 5. On day 6, a single dose of 6 μg/ml LPS (Sigma-Aldrich) was applied into the bioreactor jar in PMEC-repopulated lungs. The vascular barrier was measured and calculated by non-invasive methods as per previously described (Engler et al., 2019 (link)) for the following 24 h.
Y 27632
Manufactured by Cayman Chemical
Sourced in United States, Japan, United Kingdom
Y-27632 is a Rho-associated protein kinase (ROCK) inhibitor. It is a chemical compound used as a research tool in cell biology and biochemistry studies.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Accutase, by Innovative Cell Technologies (6 mentions)
Blebbistatin, by Cayman Chemical (4 mentions)
Geltrex, by Thermo Fisher Scientific (4 mentions)
U0126, by Cayman Chemical (3 mentions)
Sb431542, by Cayman Chemical (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
Sb202190, by Cayman Chemical (3 mentions)
Nicotinamide, by Merck Group (3 mentions)
Matrigel, by Corning (3 mentions)
U46619, by Cayman Chemical (3 mentions)
Puromycin, by Merck Group (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Sb203580, by Cayman Chemical (2 mentions)
Sp600125, by Cayman Chemical (2 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Geltrex, by Cayman Chemical (2 mentions)
Ara c, by Merck Group (2 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (2 mentions)
69 protocols using y 27632
1
Recellularization of Decellularized Lungs
2
Preparation of Bradykinin-related Compounds
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Bradykinin was purchased from Bachem (Bubendorf, Switzerland) and dissolved in acetic acid (0.1 M) to stock solutions of 10−2 M. Lys-[Des-Arg9]-Bradykinin, [Phe8Ψ(CH-NH)-Arg9]-Bradykinin, HOE-140, and R-715 were purchased from Tocris (Bristol, UK) and were dissolved in saline. Stock solutions of Lys-[Des-Arg9]-Bradykinin, [Phe8Ψ(CH-NH)-Arg9]-Bradykinin, and R-715 were 10−3 M, whereas due to its poor solubility in water, stock solutions of HOE-140 were 5 × 10−4 M. Carbachol was from Sigma-Aldrich (St. Louis, MO) and dissolved in saline to a stock solution of 2 × 10−1 M. Atropine (atropinum sulfuricum) was purchased from Egis Pharmaceutical PLC (Budapest, Hungary) and was diluted in water to a stock solution of 1.44 × 10−4 M. α,β-Methyleneadenosine 5′-triphosphate, PPADS, and Y-27632 were purchased from Cayman Chemical (Ann Arbor, MI) and all substances were dissolved in saline (α,β-meATP: 10−2 M, PPADS: 10−2 M, and Y-27632: 10−3 M). Indomethacin was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in DMSO (10−2 M stock concentration), as its aqueous solutions are quite unstable (34 (link)). NS-398 was also purchased from Sigma-Aldrich (St. Louis, MO), and DMSO was applied as solvent for preparing a 10−2 M stock solution.
3
Alkaline Phosphatase Assay for Osteogenic Differentiation
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Alkaline phosphatase (ALP) levels were determined by colorimetry using an ALP assay kit (Jiancheng Bioengineering institute, Nanjing, China). BMSCs were seeded on 6-well plates at a density of 1×10 5 cells/ well, and treated with different concentrations (0, 0.1, 0.5, 1, and 2 mM) of DMOG in osteogenic medium for 3, 7, 14, and 21 days, osteogenic medium (control), osteogenic medium containing 0.5 mM of DMOG, osteogenic medium containing 10 μM of Y-27632 (ROCK inhibitor; Cayman Chemical, Ann Arbor, MN, USA), and osteogenic medium containing 0.5 mM of DMOG and 10 μM of Y-27632 for 3, 7, 14, and 21 days. After treatment, cells were lysed with RIPA buffer (Invitrogen Inc., Carlsbad, CA, USA) at 37°C for 15 min, and treated with the chromogenic agent provided by the kit. According to previous studies and our preexperiments, 10μM of Y-27632 was used to inhibit the activity of ROCK in our experiment [31] . A standard phenol solution replaced the cell lysis solution. The blank tube contained double-distilled water instead of cell lysis solution. The blank tube was used to adjust "zero". The absorbance in each tube was measured at 520 nm. ALP levels were determined as follows: ALP=(KingU/100mL)=(OD test tube /OD Standard tube )×0.005 mg×(100 mL/0.05 mL).
4
Disrupting Cytoskeleton and Membrane Tension
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To reduce the cortical tension by disrupting the regulation of the actin cytoskeleton, cells were incubated with 10 μM cytochalasin D (037-17561, FUJIFILM), (-)-Blebbistatin (021-17041, FUJIFILM), or Y-27632 (10005583, CAYMAN) in PBS for 120 minutes at room temperature. To change the plasma membrane tension, cholesterol in the plasma membrane was removed by methyl-β-cyclodextrin (MβCD). 250 mM MβCD (332615, Sigma-Aldrich) in Milli-Q water was rotated with or without 50 mM cholesterol (C8667, Sigma-Aldrich) for 1 h, diluted to 10 mM with serum-free DMEM, and then filtered through 0.22 μm Millex-GP (SLGPR33RS, Merck). Cells were incubated with DMEM containing 10 mM MβCD or MβCD/cholesterol for 1 h at 37˚C.
To in hibit the glycolysis, cells were incubated with 1 mM 2-Deoxy-D-glucose (10722-11, Nacalai Tesque) in DMEM for 1 day at 37˚C in 5% CO2.
To in hibit the glycolysis, cells were incubated with 1 mM 2-Deoxy-D-glucose (10722-11, Nacalai Tesque) in DMEM for 1 day at 37˚C in 5% CO2.
5
Maintenance of Human iPSC Lines
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The TkDN4-M (4M) hiPSCs line was a kind gift from Dr. M Ohtsu at The Institute of Medical Science, The University of Tokyo. 4M cells were cultured as previously described with minor modifications [23 (link)]. They were maintained on mitomycin C (MMC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)-treated SNL feeder cells in hiPSCs medium (DMEM/Ham`s F12 (FUJIFILM Wako) supplemented with 20% Knockout Serum Replacement (KSR; GIBCO BRL, Palo Alto, CA, USA), 1x MEM nonessential amino acids (FUJIFILM Wako), 0.5x penicillin streptomycin(PS; FUJIFILM Wako), 55μM 2-melcaptoethanol (2ME Gibco) and 7.5μg/ml basic fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ, USA). For passage, 4M colonies were detached using CTK solution, chipped by pipetting, and seeded onto MMC-treated SNL feeder (ECACC, Salisbury, UK) in hiPSCs medium once a week. The 15M63 hiPSCs line was kindly provided from the Center for iPS Cell Research and Application of Kyoto University (Kyoto, Japan) and maintained on vitronectin (Invitrogen, CA, USA) coated dishes in StemFit. For passage, 15M63 was dissociated with accutase (Innovative Cell Technologies, San Diego, USA) by pipetting and seeded on vitronectin coated dishes in StemFit (Ajinomoto, Tokyo, Japan) supplemented with 10μM Y27632 (Cayman Chemical, Ann Arbor, MI, USA).
6
Endothelial Cell Culture and Stimulation
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HUVECs were obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS, HyClone), streptomycin (100 mg/ml; Sigma‐Aldrich, Saint Louis, Missouri, USA) and penicillin (100 units/ml; Sigma‐Aldrich) and maintained at 37 °C under a humidified atmosphere containing 5% CO2. The cells were seeded into 6‐ or 96‐well plates, stimulated by various drugs for different desired time intervals and then collected for further analysis. The drugs used to stimulate the cells were as follows: SC‐560 (COX‐1 inhibitor); NS‐398 (COX‐2 inhibitor); SC51322 (EP1 antagonist); AH6809 (EP2 antagonist); L798106 (EP3 antagonist); L‐161982 (EP4 antagonist); PD98059 (ERK inhibitor); SB203580 and SB202190 (p38 MAPK inhibitors); SP600125 (JNK inhibitor); Y27632 (ROCK inhibitor); cicaprost, PGE2, and AA (Cayman Chemical, Ann Harbor, MI, USA); TCDD (Cambridge Isotope Laboratories, Tewksbury, MA, USA).
7
Keratinocyte Culture Protocol with Cytokines
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Dispase, defined keratinocyte serum-free medium (KSFM) supplemented with keratinocyte growth factor (KGF), high glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Gibco and Invitrogen, Auckland, CA, USA). SB-216763 and Y27632 were from Cayman Chemicals (Ann Arbor, MI, USA). IL-8, IL-13, IL-17A, TGF-β1and TNF-α were purchased from PeproTech (Rocky Hill, NJ, USA). 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (St Louis, MO, USA). Fluorescein isothiocyanate (FITC)-conjugated, Cy3-conjugated and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Cocktail protease inhibitors were purchased from Roche Diagnostics (Indianapolis, IN, USA). Primary antibodies used are listed in electronic supplementary material, table S1.
8
Actomyosin Inhibitor Treatment Protocol
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For actomyosin inhibitor treatments, monolayers were prepared in a collagen-coated multi-well plate (Cellvis, P24-1.5H-N). Y-27632 (Sigma, Y0503) was used at a concentration of 10 μM for 60 minutes (min), cytochalasin-D (Sigma, C2618) at a concentration of 1.5 μM for 30 min, and okadaic acid (Cayman Chemical, 10011490) was used at a concentration of 50 nM for 12 h; an equivalent volume and time of the corresponding drug solvent was used as control for Y-27632 (water; 1:500), cytochalasin-D (DMSO; 1:3333), and okadaic acid (ethanol; 1:2500).
9
Immunofluorescence Staining of Cytoskeletal Proteins
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Anti-FN rabbit polyclonal antibody was purchased from Abcam (Cambridge, UK); Anti-F-actin rhodamine conjugated antibody, Hoechst 33342 solution, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-rabbit IgG, and Alexa Fluor 488 donkey anti-goat IgG were purchased from Thermo Fisher Scientific (Waltham, MA); Anti-elastin antibody (RA75) was purchased from Elastin Products Co. Inc. (Owensville, MI); Anti-α SMA (A2547) were purchased from Sigma (St. Louis, MO); Anti-von Willebrand factor antibody was purchased from Dako Cytomation (Glostrup, Denmark); Anti-fibrillin-1 was kindly provided by Professor T. Nakamura (Kansai Medical University, Japan); Y27632 and C3 were purchased from Cayman Chemical (Ann Arbor, MI) and Cytoskeleton, Inc. (Denver, CO), respectively.
10
Pharmacological Regulation of Cell Migration
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MEF cells were treated with 1% DMSO, 20 µM BAPTA (Cayman Chemical), Y27632 5 µM (Cayman Chemical), 50 µM CK666 (Cayman Chemical), STO-609 acetate (Biotechne, #1551), A23187 (Cayman Chemical), 4 µM Blebbistatin (Cayman Chemical), 40 uM AIP (R&D Systems #5959/1), 50 µM CAS 1090893 (Millipore, #553511), 0.1 µg/ml RhoA inhibitor-I (Cytoskeleton, Inc, #CT-04), 2 µM FK-506 (Cayman Chemical, # 10007965), 300 nM FAK14 (Cayman Chemical, #14485), 10 µM BMS-5 (Cayman Chemical, #21072), or 2 µM ML-7 (Cayman Chemical, #11801) overnight during time-lapse imaging for random cell migration or followed by immunostaining.
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