HiPSCs were generated from patient-derived primary fibroblasts as described before (van der Wal et al., 2018 (link)). Fibroblast cells from four patients with a rapid disease progression were reprogrammed using a polycistronic lentiviral vector of Oct4, Sox2, Klf4, and c-Myc (LV-OSKM). hiPSCs were cultured on γ-irradiated mouse embryonic feeder (MEF) cells with hiPSC culture medium consisting of DMEM/F12 medium (Invitrogen), 20% knock-out serum replacement (Invitrogen), 1% non-essential amino acids (Gibco), 1% penicillin/streptomycin/L-glutamine (100x, Gibco), 2 mM β-mercaptoethanol (Invitrogen) and 20 ng/ml basic fibroblast growth factor 2 (Peprotech). After gene correction, the hiPSCs were transferred to a feeder free culture with Vitronectin XF (Stem Cell) as coating and mTeSR™ Plus (Stem Cell) as media. Healthy control hiPSCs were obtained from the HIPSCI database and cultured with Vitronectin XF (Stem Cell) as coating and mTeSR™ Plus (Stem Cell) as media. Mycoplasma tests were routinely performed on all cell lines using the MycoAlert™Mycoplasma Detection Kit (Lonza) and were found negative.
Vitronectin xf
Manufactured by STEMCELL
Sourced in Canada, Germany, United States, United Kingdom
Vitronectin XF is a recombinant cell culture matrix protein. It functions as an extracellular matrix component to support the attachment and growth of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Relesr, by STEMCELL (6 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Tesr e8 medium, by STEMCELL (5 mentions)
Mtesr plus, by STEMCELL (4 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (4 mentions)
Tesr e8, by STEMCELL (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Essential 8 medium, by Thermo Fisher Scientific (3 mentions)
Pen strep, by Thermo Fisher Scientific (3 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Edta solution, by Thermo Fisher Scientific (2 mentions)
Tesr e8 media, by STEMCELL (2 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (2 mentions)
Celladhere dilution buffer, by STEMCELL (2 mentions)
236 eg 01m, by R&D Systems (2 mentions)
Gentle dissociation reagent, by STEMCELL (2 mentions)
Mtesr1 medium, by STEMCELL (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
65 protocols using vitronectin xf
1
Generation of Patient-Derived hiPSCs for Disease Modeling
2
Generation of iPSC-derived Cardiac Fibroblasts
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For the generation of iPSC-CFs a protocol developed by Zhang et al. was used13 (link). Briefly, human iPSCs were dissociated with 1 mL/well 0.5 µM EDTA solution (Invitrogen) at RT for 5 min and seeded on Vitronectin XF (StemCell Technologies) coated 6-well plates at a density of 15.000–30.000 cells/cm2 in TeSR-E8 medium (StemCell Technologies) supplemented with 5 μM ROCK inhibitor (Y-27632) (Tocris) for 24 h. Cells were cultured for 6–7 days in TeSR-E8 medium with medium changes every other day until they reached 100% confluency and differentiation started (day 0). At day 0, the medium was changed to 2.5 mL/well RPMI + B27 without insulin (Gibco) and supplemented with 12 µM CHIR99021 (Tocris) for 24 h (day 1). After day 1, the medium was changed to 2.5 mL RPMI + B27 without insulin for 24 h (day 2). Afterwards, the medium was changed to 2.5 mL/well of the CFBM medium (Table S1 ) supplemented with 75 ng/mL bFGF (StemCell Technologies). Cells were refreshed with 2 mL/well CFBM supplemented with 75 ng/mL bFGF every other day until day 20 when RNA was collected, and cells were dissociated using TrypLE Select (10x) (Thermo Fisher) for 10 min at 37 °C. After dissociation, cells were cultured in DMEM + 10% Fetal bovine serum. For the first two passages, 5 μM ROCK inhibitor was added for 24 h to help cell attachment. Cells between passage 3–6 were used for experiments.
3
Hepatocyte Differentiation from Human iPSCs
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Human iPSCs CGT-RCiB-10 (Cell & Gene Therapy Catapult) were maintained on Vitronectin XF (STEMCELL Technologies) coated Corning Costar TC‐treated six‐well plates (Sigma Aldrich) in Essential 8 Medium (Thermo Fisher Scientific) and passaged every 4 days using Gentle Cell Dissociation Reagent (STEMCELL Technologies).
Hepatocyte differentiation was carried out as previously described58 (link),59 . Silencing of human RBFOX2 was performed by transfecting 100 nM smart pool to RBFOX2 or a mock control (Horizon) with RNAimax reagent (Invitrogen) in OptiMEM (Invitrogen).
Hepatocyte differentiation was carried out as previously described58 (link),59 . Silencing of human RBFOX2 was performed by transfecting 100 nM smart pool to RBFOX2 or a mock control (Horizon) with RNAimax reagent (Invitrogen) in OptiMEM (Invitrogen).
4
Culturing and Passaging Transfected iPSCs
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Transfected iPS cells were cultured in mTeSR™ Plus (STEMCELL™ Technologies, Köln, Germany)-coated 6-well plates at 37 °C and supplemented with 1% Penicillin/Streptomycin on Vitronectin XF™ (STEMCELL™ Technologies). After thawing, cells were treated with 10 μM of rock inhibitor for 24 h and routinely split every 3–4 days on freshly coated plates using ReLeSR (STEMCELL™ Technologies).
5
Generation and Validation of hiPSC Lines
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Wild-type and HDAC9 rs9107595 risk hiPSC lines were purchased from the HiPSci Human stem cell initiative cell bank or obtained from the hiPSC core facilities at Cambridge (Supplementary Table 1 )1 ,2 and cultured in TeSR-E8 media (STEMCELL Technologies, Vancouver, BC, Canada) or E8 media (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) with Insulin-Transferrin-Selenium (Thermo Fisher Scientific, Waltham, MA, United States), Sodium Bicarbonate (Thermo Fisher Scientific, Waltham, MA, United States), and L-ascorbic acid (Merck) supplemented with FGF2 (4 ug/mL; Biochemistry Department, University of Cambridge) and TGF- β1 (1.74 ug/mL; R&D Systems, Minneapolis, MN, United States) using Vitronectin XF (STEMCELL Technologies, Vancouver, BC, Canada) as chemically defined xenofree cell culture matrix. All hiPSC lines were validated by Cambridge Biomedical Research Center iPS Core and routinely tested for presence of mycoplasma contamination by Mycoplasma Experience Ltd.
6
Maintaining and Passaging Human iPSC Cultures
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Human iPSC cultures were maintained on plates coated with 40 μL of Vitronectin XF (Stem Cell Technologies) and 1 mL of CellAdhere Dilution Buffer (Stem Cell Technologies) for 1 h in 2 mL of mTESR1 medium (mTeSR1 5 × Supplement 1:4 mTeSR1 Basal medium; Stem Cell Technologies). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and were cultured daily with mTESR1 media until reaching 80–90% confluence. The cells were typically ready for passage within 5 to 7 days. For the newly reprogrammed iPSCs (i.e., up to passage 3), colonies were mechanically passaged using a drawn-out glass Pasteur pipette to dissociate individual colonies. This method is used for passaging only desired undifferentiated iPSC colonies, and not unwanted differentiated colonies. From passage 3, iPSCs were enzymatically passaged using ReLeSR (Stem Cell Technologies). Medium was changed daily, and cells were subcultured once every 5 to 7 days.
7
Generating Alveolar Epithelial Cells from hPSCs
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Human embryonic stem cell lines hPSC1 (H9, WiCell) were cultured with mTeSR (StemCell Technologies, Inc., 85850) on Vitronectin XF (StemCell Technologies, Inc., 07180)-coated tissue culture plates. Cells were split in a ratio of 1:6 to 1:12 every 4–6 days using Gentle dissociation reagent (StemCell Technologies, Inc., 07174). Pneumocyte differentiation was induced as previously described (Riva et al., 2020 (link); White et al., 2021 (link)). Briefly, cells were collected at 70%–80% confluency and approximately, 2 million cells per 10 cm2 were plated on 12-well Vitronectin-coated tissue culture plates in mTeSR. The next day, definitive endoderm differentiation was induced as previously described (Jacob et al., 2019 (link)). Cells were split after 4 days and further induced to differentiate based on an adapted alveolar differentiation protocol (Ghaedi et al., 2013 (link)) in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies, 31980030) supplemented with 10% FBS (Sigma, F4135), 2 mM L-glutamine (Life Technologies 25030081), 0.5 μM all-trans-retinoic acid (Sigma, R2626), 10 ng/mL FGF-10 (R&D Systems, 345-FG-025), 10 ng/mL EGF (R&D Systems, 236-EG-01M), 100 ng/mL Wnt3a (R&D Systems, 5036-WN-010), 10 ng/mL KGF (R&D Systems, 251-KG-050) and 5 ng/mL BMP-4 (R&D Systems, 314-BP-010). Viral infections were performed on day 11 of differentiation.
8
Expansion and Maintenance of hiPSCs
9
Maintaining Human Induced Pluripotent Stem Cells
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The human induced pluripotent stem cell (hiPSC) line HPSI1213i-babk_2 was obtained from the Wellcome Trust Sanger Institute in Cambridge, UK and was used for all experiments. hiPSCs were maintained in Essential 8™ Flex Medium (Gibco) in 6-well tissue culture plates coated with 10 μg/mL Vitronectin XF (StemCell Technologies) in a 37 °C incubator containing 95% air 5% CO2. hiPSCs were passaged at 70% confluency using ReLeSr™ (StemCell Technologies). Essential 8™ Flex Medium was supplemented with Y-27632, a specific Rho-associated, coiled-coil containing protein kinase (ROCK) Inhibitor (10 μM) (Tocris) for 24 h following passaging.
10
Platelet Adhesion under Shear Stress
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Platelet adhesion under varying shear rates was assessed in ibidi µ-slides VI0.1 (Ibidi, Gräfelfing, Germany). Slides were coated either with fibrin(ogen) (100 µg/ml in TBS, Sigma-Aldrich, Taufkirchen, Germany) or Vitronectin XF (25 µg/ml in TBS, STEMCELL Technologies, Köln, Germany) together with collagen-related peptide (CRP, 0.5 µg/ml, kindly provided by Prof. Siess, LMU, Munich) at 4°C overnight. Blood collected from wild-type mice was mixed with 1/2 volume of heparin (20 U/ml in TBS) and perfused through fibrinogen/CRP and vitronectin/CRP-coated flow chambers at defined flow rates resulting in 1, 5, 10 or 50 dyne/cm2 wall shear stress for 4 min using a PHD ULTRA pump (Harvard Apparatus, Holliston, MA, USA). Subsequently, flow chambers were washed by perfusing Tyrode’s buffer (136 mM NaCl, 0.43 mM NaH2PO4, 2.7 mM KCl, 12mM NaHCO3, 2 µM CaCl2, 1 µM MgCl2, 5 mM HEPES, 0.1% glucose, 0.35% BSA; pH 7.35) at the respective flow rate for 20 min and adherent platelets were stained with CellTrace CFSE dye (Thermo Fisher Scientific, Darmstadt, Germany). Images were acquired with an Evos M7000 life cell microscope (Thermo Fisher Scientific) and surface coverage was quantified using ImageJ software.
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