At day 0 of time courses, H9-H2BmCherry and EGFP-mEpiS cells were washed with PBS (Thermo Fisher Scientific, USA), treated with TrypLE (Thermo Fisher Scientific, USA) for singularization, and resuspended in a simple neural differentiation medium consisting of DF3S (DMEM/F-12, L-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14 μg/L), and NaHCO3 (543 mg/L), Thermo Fisher Scientific, USA), 1XN2 supplement (Thermo Fisher Scientific, USA), 1XB27 supplement (Thermo Fisher Scientific, USA), and 100ng/mL of mNoggin (R&D Systems, USA). To aid cell survival, 10 μM Y27632 ROCK inhibitor (Tocris Bioscience, UK) was added on day 0, and cells were mixed at the indicate mouse-human ratios and seeded into Matrigel-coated 12-well plates at 2.5X105 cells/well in triplicate. Media in all wells was replaced with fresh neural differentiation media (without ROCK inhibitor) every day for the 42 days of differentiation. When cells become over-confluent cells were split 1:3 or 1:6 by EDTA-treatment to avoid disrupting cell-cell interactions.
Rock inhibitor y 27632
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
ROCK inhibitor Y-27632 is a small molecule compound that selectively inhibits the Rho-associated protein kinase (ROCK) enzymes. ROCK plays a key role in regulating cell cytoskeleton and cell contractility. Y-27632 inhibits ROCK activity, leading to modulation of cytoskeletal dynamics and cell behavior.
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Lab products found in correlation
Matrigel, by Corning (9 mentions)
Chir99021, by Bio-Techne (9 mentions)
Tryple select, by Thermo Fisher Scientific (6 mentions)
Tryple, by Thermo Fisher Scientific (5 mentions)
Basic fgf, by Thermo Fisher Scientific (5 mentions)
Stemfit basic04 medium, by Ajinomoto (5 mentions)
Rpmi b27 without insulin, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Accutase, by Thermo Fisher Scientific (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
Relesr, by STEMCELL (4 mentions)
Mtesr1 medium, by STEMCELL (4 mentions)
Matrigel, by BD (4 mentions)
Gfr matrigel, by BD (4 mentions)
Tryple express, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
E8 medium, by Thermo Fisher Scientific (3 mentions)
77 protocols using rock inhibitor y 27632
1
Neural Differentiation of Human and Mouse Cells
2
Protein Detection: Antibodies and Reagents
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A summary of the primary antibodies used in this study can be found in SI Appendix, Table S1 . The following secondary antibodies conjugated to horseradish peroxidase were used for immunoblotting: RRID:AB_2099233, RRID:AB_330924, and RRID:AB_228395. Immunofluorescence experiments used the following secondary antibodies: RRID:AB_2535794 and RRID:AB_141637. The following chemicals were used: Y-27632 Rock inhibitor (Tocris Bioscience), MLi-2 (Abcam #254528), LRRK2-IN-1 (Tocris Bioscience # 4273), and EDTA (Invitrogen #15575-038).
3
Dose-Response Evaluation of Lapatinib and Paclitaxel in Breast Cancer Cell Lines and Organoids
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MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were seeded in DMEM-10% FBS in a 96-well cell culture plate with 10,000 cells per well. After cell attachment, various concentrations of lapatinib (Selleckchem) and paclitaxel (Selleckchem) were used to treat the cells. Following 48-h treatments, MTS (Sigma) was added to the wells according to the manufacturer’s instructions to obtain dose–response curve. MC-BR-BTY-0019 and MC-BR-BTY-0006 organoids were grown from corresponding PDX tumors freshly harvested from mice and cultured in MEF media with 10,000 cells per well in nanoculture plates. Briefly, PDX tumors were collected when the tumor grew to ~10–20 mm diameter. Primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described7 (link). A single-cell suspension of primary breast cancer cells was initially cultured in MEF media (DMEM with 10% FBS supplemented with glutamax, MEM NEAA, sodium pyruvate, and 5 μM Y-27632 ROCK inhibitor (Tocris)). ROCK inhibitor was removed from media by changing media one week before drug treatment. Cells were treated with various concentrations of lapatinib or paclitaxel for 5 days. 3D cell TiterGlo kit (Promega) was used to measure 3D culture viability according to the manufacturer’s instructions to obtain dose–response curves.
4
Differentiation of Neural Stem Cells
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NAG-NSCs were grown until confluent and then detached using TrypLE Express Enzyme (Gibco) and plated onto a T-650 flask coated with 50 μg/ml poly-D-lysine (Sigma-Aldrich) and 10 μg/ml mouse laminin (ThermoFisher). Cells were plated at a density of 0.7 × 108–1.0 × 108 cells/flasks in Neuron Differentiation Media supplemented 100 μg/ml Cumate (System Biosciences), 1 μM PD0332991 cell cycle inhibitor (Tocris), and 10 μM Y27632 Rock inhibitor (Tocris). Cells were differentiated for 5–7 days with one half volume differentiation media changed every 3 days.
5
Culturing Human and Mouse Pluripotent Cells
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Human ES and mEpiS cells were cultured and passaged as previously reported[1 (link)]. Briefly, H9 cells were cultured in E8 Medium (Thermo Fisher Scientific, USA) on Matrigel-coated plates and split every 2–3 days with EDTA. To easily identify human from mouse cells, H9 cells were electroporated with a selectable PiggyBAC-inserted plasmid expressing nuclear-localized H2B-mCherry driven by the EF1α promoter, and clonally expanded.
EGFP-expressing mEpiS cells derived from C57BL/6-Tg(CAG-EGFP)1Osb/J (JAX Stock No. 003291) mice and cultured as previously described[1 (link),16 (link),18 (link)]. Cell were maintained on low passage MEFs and cultured in DMEM/F12 medium (Thermo Fisher Scientific, USA) supplemented with 20% Knockout serum replacement (Thermo Fisher Scientific, USA), 0,18 mM B-mercaptoethanol (Sigma, USA), 1Xnon-essential amino acids (Thermo Fisher Scientific, USA), 2 mM L-glutamine (Sigma, USA), 7.5 ng/mL activin A (R&D Systems, USA), and 5ng/mL bFGF (R&D Systems, USA). Cells were passaged by adding TrypLE (Thermo Fisher Scientific, USA) and seeding onto fresh MEFs with 10 μM Y27632 ROCK inhibitor overnight to increase cell survival (Tocris Bioscience, UK).
EGFP-expressing mEpiS cells derived from C57BL/6-Tg(CAG-EGFP)1Osb/J (JAX Stock No. 003291) mice and cultured as previously described[1 (link),16 (link),18 (link)]. Cell were maintained on low passage MEFs and cultured in DMEM/F12 medium (Thermo Fisher Scientific, USA) supplemented with 20% Knockout serum replacement (Thermo Fisher Scientific, USA), 0,18 mM B-mercaptoethanol (Sigma, USA), 1Xnon-essential amino acids (Thermo Fisher Scientific, USA), 2 mM L-glutamine (Sigma, USA), 7.5 ng/mL activin A (R&D Systems, USA), and 5ng/mL bFGF (R&D Systems, USA). Cells were passaged by adding TrypLE (Thermo Fisher Scientific, USA) and seeding onto fresh MEFs with 10 μM Y27632 ROCK inhibitor overnight to increase cell survival (Tocris Bioscience, UK).
6
Generating LAMP1-GFP Lentiviral Construct
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LAMP1-GFP was amplified using PCR (using primers listed in Supplemental Table S2) from Addgene plasmid #34831 and then cloned into lentiviral vector FUGW (Addgene plasmid #14883) from which the GFP fragment had been excised by digesting the vector with BamHI and EcoRI. FUGW was a gift from David Baltimore, California Institute of Technology (Addgene plasmid #14883; http://n2t.net/addgene: 14883; RRID:Addgene_14883). LAMP1-mGFP was a gift from Esteban Dell’Angelica, UCLA, Trono (Addgene plasmid #34831; http://n2t.net/addgene: 34831; RRID:Addgene_34831).
HEK 293 FT cells were transfected with the newly made LAMP1-GFP lentiviral vector, along with the psPAX2 (Addgene #12260) and PCMV (Addgene #8454) packaging plasmids. pCMV-VSV-G was a gift from Bob Weinberg, Massachusetts Institute of Technology (MIT) (Addgene plasmid #8454;http://n2t.net/addgene:8454 ; RRID:Addgene_8454). psPAX2 was a gift from Didier Trono (Addgene plasmid #12260; http://n2t.net/addgene:12260 ; RRID:Addgene_12260).
The supernatant from these cells containing the virus was collected, concentrated, and then diluted into E8 medium containing the Y-27632 ROCK inhibitor (Tocris). This was added to freshly split iPSCs (1.5 million cells in one well of a six-well plate). The media on the cells was changed after 48 h. These cells were then expanded, and LAMP1-GFP–expressing cells were selected by FACS.
HEK 293 FT cells were transfected with the newly made LAMP1-GFP lentiviral vector, along with the psPAX2 (Addgene #12260) and PCMV (Addgene #8454) packaging plasmids. pCMV-VSV-G was a gift from Bob Weinberg, Massachusetts Institute of Technology (MIT) (Addgene plasmid #8454;
The supernatant from these cells containing the virus was collected, concentrated, and then diluted into E8 medium containing the Y-27632 ROCK inhibitor (Tocris). This was added to freshly split iPSCs (1.5 million cells in one well of a six-well plate). The media on the cells was changed after 48 h. These cells were then expanded, and LAMP1-GFP–expressing cells were selected by FACS.
7
Deriving and Culturing hPSC-Cardiomyocytes
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A REBL-PAT hPSC line was derived from a skin punch biopsy from a male subject. Procedures of isolation, culture, differentiation and dissociation are described elsewhere [32 (link)]. Early hPSC-CMs were seeded on the photo-cured gels. Dissociation of cells took place 6–8 weeks after the differentiation process and cells were seeded on hydrogels at an approximate concentration of 2 million cells mL−1 in basal RPMI medium (Life Technologies #11875093) supplemented with B27 (Life Technologies #17504044, Carlsbad, CA, USA), Y-27632 ROCK inhibitor (20 µM; Tocris #1254) and 10% foetal bovine serum (Sigma-Aldrich). The medium was changed after 24 h to RPMI/B27 medium.
8
Maintaining Human Induced Pluripotent Stem Cells
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The wild-type (K3) line of hiPSC was established previously by Si-Tayeb et al. [27 (link)] and was provided for our studies. The cell line was maintained in StemFlex medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) on Vitronectin-coated dishes at 37 °C with 5% CO2. The culture medium was replaced every 2 days and hiPSCs were passaged with Accutase (Thermo Fisher) every 4–6 days. A 10 μM quantity of Y-27632 ROCK inhibitor (Tocris) was added to the culture media for 24 h after each passage. Cell passages between No. 50 and No. 60 were used for the experiments.
9
Directed Differentiation of ESCs into Definitive Endoderm
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For embryoid body formation, ES cells were resuspended in hanging drops comprising IMDM medium (Life Technologies 12440-His 053) supplemented with 10 % Hyclone FBS, 1 % L-Glutamine, 1 % Penicillin/Streptomycin, 5 % Protein Free Hybridoma Medium II (PFMHII) (Life Technologies 12040–077), 0.5 mM Ascorbic Acid (Sigma A4403), 4.5×10−4 M Monothioglycerol (Sigma M6145), and 200 μg/mL Transferrin (Sigma T8158). 1 day later, embryoid bodies were re-plated into non-coated petri dishes and monitored over the course of 3–7 days for fluorescence. For directed differentiation into definitive endoderm using growth factors, cells were incubated in standard ES cell medium (as described above) supplemented with 1 % N-2 (Life Technologies 17502–048), 2 % B-27 (Life Technologies 17504044), and 2.5 μM Y-27632 ROCK inhibitor (Tocris 1254) for 24 h. After 24 h, media was changed to standard ESC media supplemented with 50 ng/mL E. coli Activin A (Pepro-Tech, 120-14E) and 5 nM GSK3 inhibitor XV (Calbiochem 361558). After 24 h, media was changed to standard ESC media supplemented with 2 μM Dorsomorphin (Sigma P5499) and 50 ng/mL E. coli Activin A (Pepro-Tech, 120-14E), and changed daily for 1–3 days. Transient overexpression of GATA4-mCherry was performed using Gata6H2B-Venus/+;ColA1TetO-Gata4-mCherry/+;R26M2rtTA/+ ES cells [50 ] incubated with 1 mg/mL Doxycycline (Sigma D9891), replaced daily for 48 h.
10
Human iPSC Arsenic Exposure Protocol
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Human iPS cells (DYS0100, ATCC, Manassas, VA, USA) were cultured in mTeSR1 medium (StemCell, Vancouver, BC, Canada) on 6-well plates coated with Matrigel (Corning, Corning, NY, USA). Cells were maintained in a humidified incubator at 37 °C and 5% CO2 and medium was changed daily.
To determine appropriate exposure concentrations, on Day -1, human iPS cells were dissociated with ReLeSR (StemCell) and plated on 6-well Matrigel-coated plates (2 × 105 cells/well) with mTeSR1 medium containing 10 µM Y-27632 ROCK inhibitor (Tocris, Bristol, UK). From Day 0 to Day 6, cells were cultured in mTeSR1 medium containing 0, 0.1, 0.25, or 0.5 µM arsenic as sodium arsenite (Sigma Aldrich, St. Louis, MO, USA), with daily medium changes. At day 6 (D6), cells were collected for flow cytometry or stored at −80 °C in TRIzol (Sigma Aldrich) for subsequent RNA extraction. Control and arsenic-exposed samples were cultured as independent replicates (n = 3–6 for each treatment).
To determine appropriate exposure concentrations, on Day -1, human iPS cells were dissociated with ReLeSR (StemCell) and plated on 6-well Matrigel-coated plates (2 × 105 cells/well) with mTeSR1 medium containing 10 µM Y-27632 ROCK inhibitor (Tocris, Bristol, UK). From Day 0 to Day 6, cells were cultured in mTeSR1 medium containing 0, 0.1, 0.25, or 0.5 µM arsenic as sodium arsenite (Sigma Aldrich, St. Louis, MO, USA), with daily medium changes. At day 6 (D6), cells were collected for flow cytometry or stored at −80 °C in TRIzol (Sigma Aldrich) for subsequent RNA extraction. Control and arsenic-exposed samples were cultured as independent replicates (n = 3–6 for each treatment).
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