Y0503

Manufactured by Merck Group

Y0503 is a high-performance laboratory equipment designed for precise and efficient sample preparation. It features advanced technology and a compact design to optimize laboratory workflows. The core function of Y0503 is to facilitate the handling and processing of various samples, ensuring accurate and reliable results.

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Lab products found in correlation

4 protocols using y0503

Differentiation of iPSCs into Functional Neurons

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NPC1 iPSCs were differentiated into neural stem cells (NSCs) by using a kit from Life Technologies. Briefly, iPSCs were digested with Dispase and re-seeded onto Geltrex-coated plate at 20% confluence. After cells attached, the medium was changed to Induction Medium containing the Neurobasal Medium plus Neural Induction Supplement (A15640SA, Life Technologies). At day 7 of neural induction, the initial NSCs were dissociated with Accutase and plated for further expansion in Neural Expansion Medium containing Neurobasal Medium and Advanced DMEM/F12 with 1x Neural Induction Supplement. NSCs were characterized by staining with antibodies against nestin and Sox2 that showed 99% positive cells with both neural markers.
For neuronal differentiation, dissociated NSCs were cultured on poly-L-ornithine and laminin coated 96-well plates in the Induction Medium with 5 μM Y27632, a ROCK inhibitor (Y0503, Sigma-Aldrich) for one day. The medium was changed to a differentiation medium containing Neurobasal Medium, 1x B27 (17504-044, Life Technologies), 1x glutamax (35050, Life Technologies), 200μM L-ascorbic acid (A8960, Sigma-Aldrich), 1μM cAMP (A6885, Sigma-Aldrich), 10ng/ml BDNF (10908-010, Life Technologies) and 10ng/ml GDNF (PHC7044, Life Technologies) that was changed every two days for 12 days.^{20 (link)}

Yu D., Swaroop M., Wang M., Baxa U., Yang R., Yan Y., Coksaygan T., DeTolla L., Marugan J.J., Austin C.P., McKew J.C., Gong D.W, & Zheng W. (2014). Niemann-Pick disease type C: induced pluripotent stem cell-derived neuronal cells for modeling neural disease and evaluating drug efficacy. Journal of biomolecular screening, 19(8), 1164-1173.

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Quantifying Cell Migration Dynamics

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Cells were plated at a concentration of 5 × 10⁵ cells per well in a six-well plate in the presence of ethanol (−dox) or 1 μg ml⁻¹ dox (+dox) 24 h before assay. Cells were trypsinised, counted, and ≈5 × 10⁵ cells were then incubated with Dic16 dye (2.5 μM) for 30 min at 37 °C and 5% CO₂. Dic16-labelled cells (5 × 10⁴ cells per well) were seeded on fibronectin (5 μg ml⁻¹; Sigma-Aldrich, F1141) coated 96-well plates fitted with stoppers (Platypus Technologies, CMA1.101) together with ethanol (−dox) or 1 μg ml⁻¹ dox (+dox). Cells were incubated overnight in a humidity chamber at 37 °C and 5% CO₂ before the removal of the stoppers. For analysis of cell migration following inhibition of cell contraction, cells were incubated with DMEM+10% Tet-free FBS alone (control), or supplemented with the inactive enantiomer of blebbistatin [(+)-blebbistatin] (100 μM; Merk Millipore, 203392), blebbistatin [(±)-blebbistatin] (100 μM; Merk Millipore, 203390) or the ROCK inhibitor Y27632 (10 μM; Sigma-Aldrich, Y0503) for 24 h post stopper removal. Fluorescence images were taken using the low light microscope system (× 4 and × 5 magnification) at 0 and 24 h upon removal of stoppers and cell migration was quantified as outlined in Supplementary Methods.

Marei H., Carpy A., Woroniuk A., Vennin C., White G., Timpson P., Macek B, & Malliri A. (2016). Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration. Nature Communications, 7, 10664.

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Establishing and Treating Cell Lines

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Human HT-1080 fibrosarcoma, A431 epidermoid carcinoma, MCF-7 breast cancer, and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/liter), l-glutamine, and sodium pyruvate (11995073, Gibco). Medium was supplemented with 10% heat-inactivated FBS (16140071, Gibco) and 1% penicillin-streptomycin (10,000 U/ml; 15140122, Gibco). Cells were maintained at 37°C with 5% CO₂. A431 cells expressing GFP-E-cad were a gift from V. Bruntons (EH4 2XR, University of Edinburgh, UK) (63 (link)). In select experiments, cells were treated with the following pharmacological agents: Y-27632 (20 μM; Y0503, Sigma-Aldrich), ionomycin (0.5 and 1 μM; I0634, Sigma-Aldrich), blebbistatin (50 μM; B0560, Sigma-Aldrich), Rho inhibitor I (1 μg/ml; CT04-A, Cytoskeleton Inc.), LPA (50 μM; L7260, Sigma-Aldrich), paclitaxel (taxol equivalent) (1 μM; P3456, Thermo Fisher Scientific), colchicine (125 μM; C9754, Sigma-Aldrich), and importazole (50 μM; SML0341, Sigma-Aldrich).

Law R.A., Kiepas A., Desta H.E., Perez Ipiña E., Parlani M., Lee S.J., Yankaskas C.L., Zhao R., Mistriotis P., Wang N., Gu Z., Kalab P., Friedl P., Camley B.A, & Konstantopoulos K. (2023). Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement. Science Advances, 9(2), eabq6480.

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Pharmacological Inhibition of Signaling Pathways

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For pharmacological inhibition, cells were treated using the following concentration of drugs prior to sample fixation or laser ablation experiment: sodium orthovanadate (S6508, Sigma) 100 μM, RK682 (RK2033, Sigma) 10 μg/ml; ALLN (ab141445, Abcam) 10 and 50 μM, ALLM (ab141446, Abcam) 10 and 50 μM, nocodazole (M1404, Sigma) 10 μM, and Y-27632 (Y0503, Sigma) 10 μM.
While sodium orthovanadate is a generic alkaline and tyrosine phosphatase inhibitor, RK682 is a specific and non-competitive inhibitor of protein tyrosine phosphatase with confirmed low micromolar inhibitory activity against protein tyrosine phosphatases CD45 and PTP1B, and dual-specificity phosphatases VHR, CDC-25A-B-C (Hamaguchi et al., 1995 (link)); nevertheless, CD45 and VHR genes are not expressed in MDCK and EpH4 cells, as indicated by RNAseq data (Supplementary Table 1), while inhibition of CD25 requires longer incubation period (20 h) with respect to what we have used in the current paper (1 h).
ALLN and ALLM are inhibitors for calpains, with high binding efficiency for calpain-1 and calpain-2, respectively. ALLN has been reported to inhibit the calpain/PTP1B axis in endothelial cells (Zhang et al., 2017 (link)).

Joy-Immediato M., Ramirez M.J., Cerda M., Toyama Y., Ravasio A., Kanchanawong P, & Bertocchi C. (2021). Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions. Frontiers in Cell and Developmental Biology, 9, 669086.

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