The vials of hiNPCs were quickly thawed in the palms of the hand and each vial containing 4 × 106 cells was directly diluted in 10 mL of the respective neural progenitor medium with 10 µM Y-27632 (#HB2297, Hello Bio, Bristol, UK). After centrifugation (300× g, 5 min), the cell pellet was resuspended in the respective neural progenitor medium with 10 µM Y-27632 (#HB2297, Hello Bio, Bristol, UK). The cells of one frozen cryovial were divided into three wells of a coated 6-well plate. The medium was replaced daily without the addition of Y-27632 (2D-NIM and GNEIB protocol) or with 10 µM Y-27632 (Stemdiff protocol).
Y 27632
Manufactured by Hello Bio
Sourced in United Kingdom, United States
Y-27632 is a Rho-associated protein kinase (ROCK) inhibitor. It is a small molecule that selectively inhibits the activity of the ROCK enzymes. ROCK is involved in various cellular processes, including cell adhesion, motility, and contractility.
Automatically generated - may contain errors
Lab products found in correlation
Accutase, by STEMCELL (2 mentions)
Ouabain, by Bio-Techne (1 mentions)
S nitro blebbistatin, by Cayman Chemical (1 mentions)
Plan apochromatic 100 silicone, by Nikon (1 mentions)
Calyculin a, by Cayman Chemical (1 mentions)
Cytochalasin d, by Bio-Techne (1 mentions)
Methylstat, by Merck Group (1 mentions)
Sb525334, by Selleck Chemicals (1 mentions)
Stemdiff smadi neural induction kit, by STEMCELL (1 mentions)
Stemdiff neural progenitor freezing medium, by STEMCELL (1 mentions)
Penicillin streptomycin, by PAN Biotech (1 mentions)
6 protocols using y 27632
1
Thawing and Culturing Human Neural Progenitor Cells
2
Differentiation of hiPSCs into hiNPCs
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The Stemdiff protocol was performed using the STEMdiff™ SMADi Neural Induction Kit (#08581, Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol with minor modifications. Briefly, the hiPSC colonies were singularized as described above and resuspended in STEMdiff™ Neural Induction Medium supplemented with STEMdiff™ SMADi Neural Induction Supplement and 1% Penicillin/Streptomycin (#P06-07100, PAN-Biotech, Aidenbach, Germany). The cells were transferred to a PLO (15 µg/mL; #P4957, Sigma-Aldrich, St. Louis, MO, USA)-laminin (10 µg/mL; #L2020, Sigma-Aldrich, St. Louis, MO, USA)-coated 6-well plate with 10 µM Y-27632 (only for the first 24 h after passaging; #HB2297, Hello Bio, Bristol, UK). The cells were passaged on day 6 using Accutase (#07920, Stemcell Technologies, Vancouver, BC, Canada) and transferred to a new PLO-laminin-coated 6-well plate with a cell density of 1.5 × 106 cells per well. On day 12, the hiNPCs were singularized with Accutase and frozen in STEMdiff™ Neural Progenitor Freezing Medium (#05838, Stemcell Technologies, Vancouver, BC, Canada) containing 10 µM Y-27632 (#HB2297, Hello Bio, Bristol, UK).
3
Hiinduced Neural Progenitor Cell Spheres
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On day 4 after thawing, the hiNPCs were singularized with Accutase for 10 min at 37 °C and 5% CO2 (#07920, Stemcell Technologies, Vancouver, BC, Canada) and centrifuged (300× g, 10 min). After the cell pellet was resuspended in the respective neural progenitor medium with 10 µM Y-27632 (#HB2297, Hello Bio, Bristol, UK), 2 × 106 cells were transferred into one well of a new 6-well plate (#83.3920, Sarstedt, Nürmbrecht, Germany) in 4 mL medium. Sphere formation took place in a gyrical shaking incubator (#LT-XC, Kuhner Shaker GmbH, Basel, Switzerland) at 140 rpm, 12.5 mm diameter, 37 °C, 5% CO2, and 85% humidity for 7 days. On day 7, to equalize the size of the hiNPC spheres, they were chopped to 250 µm (McIlwain tissue chopper, Mickle Laboratory Engineering Co. LTD., Guildford, UK) as described previously [71 (link)].
4
Actin and Ezrin Inhibitor Treatments
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Cells were treated with the actin inhibitor cytochalasin-D (5 µM; Tocris), the ezrin inhibitor NSC668394 (50 µM; Calbiochem), Y27632 (25 µM; Hello Bio), calyculin A (10 nM; Cayman Chemical), Ouabain (30 nM; Tocris), and s-nitro-Blebbistatin 25 µM; Cayman Chemical). For cytochalasin-D and calyculin A treatment, an 18-slice Z-stack (140 nm step size) was acquired every minute for 5 min using a Plan Apochromatic 100× silicone oil immersion objective (Nikon; NA 1.35) as described above. After 5 min, a solution of cytochalasin-D dissolved in imaging media was injected into the well and the cells were imaged for another 15 min posttreatment. For NSC668394, Y27632, Ouabain, and S-nitro- Blebbistatin, cells were treated immediately before imaging and an 18-slice Z-stack (192.5 nm step size) was acquired every hour for 17 h using a Plan Apochromatic 40× air objective (Nikon; NA 0.95) and a 2× zoom lens (80× total magnification). Each drug treatment experiment was repeated in three separate wells, and multiple fields of view were collected per well.
5
Cytoskeletal and Chromatin Modulation
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For cytoskeletal inhibition, cells in suspension were incubated with 2 µm cytochalasin D (actin disruption) (Fischer Scientific), 1 µm nocodazole (microtubule disruption) (Sigma Aldrich), and 10–20 µM Y27632 (ROCK inhibition) (Hellobio, Princeton, NJ, USA) for 30 min–1 h. For chromatin compaction/decompaction, cells were pre‐treated with histone demethylase inhibitor (Methylstat, 5 µm , 48 h incubation) (Sigma Aldrich, St. Louis, MO, USA) and HDACi VPA (2 mm , 24 h incubation) (Sigma Aldrich), respectively. At least N = 2 replicates were performed for each drug condition.
6
TGFβ-Induced Migration Assay in A549 Cells
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A549 cells were seeded at a density of 5.0 × 103 cells per microwell in a 2% agarose dish. After 24 h, the medium was changed into basal DMEM/F12 medium supplemented with 10 ng/mL TGFβ in presence or absence of DMSO, 10 µM SB525334 (TGFβRI inhibitor, Selleckchem, Houston, TX, USA), or 20 µM Y-27632 (ROCK inhibitor, Hello Bio, Princeton, NJ, USA). After 48 h, 12 total spheroids were transferred to a gelatin-coated dish with the aforementioned treatment media. These were then allowed to migrate for 24 h and the migratory parameters were assessed via phase-contrast microscopy. Area of migration and normalized migration area were calculated based on a previously published paper [16 (link)].
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