Human iPSCs were routinely cultured in mTeSR1 on Matrigel (growth factor reduced) (BD Biosciences, Bedford, MA, USA)-coated plates for the TKDA3-4 iPSC clone and in StemFit (AK03N, Ajinomoto, Tokyo, Japan) on Laminin-511 E8 (Nippi, Tokyo, Japan)-coated dishes for the clinical xeno-free iPSC clones including D2, 1383D6, 1231A3, and Ff06. To generate definitive endoderm cells, we plated monolayers of pluripotent cells harvested using Accutase (Millipore, Billerica, MA, USA) on 6-well plates (Corning Costar #3516) (Corning, NY, USA) pre-coated with 1:30 diluted Matrigel (growth factor reduced; BD Bioscience) or 0.5 μg/cm2 Laminin-511 E8 (Nippi) at a density of 6 × 105 cells per well with 100 ng/mL activin A (R&D Systems, Minneapolis, MN, USA), 50 ng/mL Wnt3a, and 10 μM Rock inhibitor Y27632 in RPMI/B27 (Invitrogen, Carlsbad, CA, USA) medium for 1 day. Differentiation was initiated by culture with 100 ng/mL activin A and 50 ng/mL Wnt3a in RPMI/B27 medium for 1 day under 5% CO2, followed by 2 days with 100 ng/mL activin A, 0.5 ng/mL BMP4 (Peprotech, Rocky Hill, NJ, USA), 5 ng/mL FGF2 (Invitrogen), and 10 ng/mL VEGF (Gibco, Carlsbad, CA, USA) in RPMI/B27 under 5% CO2, then followed by another 2 days with 100 ng/mL activin A, 0.5 ng/mL BMP4 (Peprotech), 5 ng/mL FGF2 (Invitrogen), and 10 ng/mL VEGF (Gibco) in SFD-based medium under 5% CO2.
Laminin 511 e8
Manufactured by Nippi
Sourced in Japan
Laminin-511 E8 is a recombinant protein that is a truncated form of the laminin-511 protein. Laminin-511 is a key component of the extracellular matrix and plays a role in cell adhesion, migration, and differentiation. The E8 fragment retains the essential cell-binding domains of the full-length protein.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi b27, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Stemfit ak03n, by Ajinomoto (1 mentions)
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
Stemfit ak01, by Ajinomoto (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Activin a, by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Activin a, by R&D Systems (1 mentions)
Matrigel, by BD (1 mentions)
Stemfit, by Ajinomoto (1 mentions)
Laminin 521, by BioLamina (1 mentions)
Matrigel, by Corning (1 mentions)
A83 01, by Bio-Techne (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Transwell insert, by Corning (1 mentions)
6 protocols using laminin 511 e8
1
Definitive Endoderm Differentiation from iPSCs
2
Reprogramming HDFs and PBMCs into iPSCs
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HDFs or PBMCs from healthy control subjects and HMSN-P patients were reprogrammed by introducing the episomal vectors with OCT3/4, SOX2, KLF4, L-MYC, LIN28, and p53 carboxy-terminal dominant-negative fragment as previously described [34 (link)]. Established iPSCs were grown under feeder-free conditions on laminin-511 E8 (Nippi, Tokyo, Japan) -coated plates with StemFit AK03N (Ajinomoto, Tokyo, Japan) [35 ]. Another human iPSC clone, TIG107 [17 (link)], was added to the control iPSC group.
3
Generating iPSCs from ALS Patients
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iPSCs were generated from skin fibroblasts or peripheral blood mononuclear cells of sporadic ALS patients using episomal vectors (Sox2, Klf4, Oct3/4, L-Myc, Lin28, and p53-short hairpin RNA) as reported previously.21 (link),22 (link) Established iPSCs were cultured on an SNL feeder layer with human iPSC medium (primate embryonic stem cell medium; ReproCELL, Yokohama, Japan) supplemented with 4 ng/mL basic fibroblast growth factor (Wako Chemicals, Osaka, Japan) and penicillin/streptomycin or cultured under feeder-free conditions on laminin-511-E8 (Nippi, Tokyo, Japan)-coated plates with StemFit AK01 (Ajinomoto, Tokyo, Japan). The information on iPSC clones is listed in Table S3 . Karyotyping was performed by Nihon Gene Research Laboratories (Sendai, Japan).
4
Extracellular Matrix Coatings for Cell Culture
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All ECM coating solutions were prepared as recommended by the manufacturers. Wells were coated with laminin511-E8 (LN511; Nippi, Tokyo, Japan), laminin-521 (LN521; BioLamina, Sundbyberg, Sweden), recombinant truncated human vitronectin (VTN; Thermo Fisher Scientific, Waltham, MA, USA), or Matrigel® (MG; Corning, New York, NY, USA) at concentrations of 0.25–1 μg/ml overnight at 4 °C prior to cell seeding. Matrigel was prepared by diluting the matrix solution 1:100 in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12). The protein concentration of matrix solution was determined as 159 μg/ml by BCA assay, and was coated at concentration of 8.3–33.1 μg/cm2 overnight at 4 °C prior to cell seeding. Volumes of coating solution were adjusted according to the growth area of the culture vessels used.
5
Differentiation and Maintenance of hiPSC-Derived Intestinal Epithelial Cells
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The hiPSC-IECs were seeded on laminin511-E8 (Nippi, Tokyo) coated plates or Transwell inserts (Corning, Corning) at a high density of 2–3 × 105 cells/cm2 in advanced DMEM/F12 supplemented with 2 mM L-glutamine, 15 mM HEPES, 2% B27 supplement, 1% (v/v) N2 supplement (Thermo Fisher Scientific, Waltham), 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM N-acetylcysteine (Sigma-Aldrich, Saint Louis), 2.5 μg/mL amphotericin B (infrequent) (Sigma-Aldrich, Saint Louis), 10 nM human gastrin Ι (Peptide Institute, Osaka), 500 nM A-83-01 (Tocris Bioscience, Minneapolis), 10 μM Y27632 (only on the day of seeding) and 500 ng/mL recombinant human R-Spondin1, 100 ng/mL recombinant human Noggin and 100 ng/mL recombinant human EGF. For long-term culture, the hiPSC-IECs were embedded in Matrigel at 5,000-10,000 cells on 24-well culture plates or 100-mm culture dishes. After the Matrigel had solidified, the culture medium described above was overlaid and replaced every 3 or 4 days.
6
Isolation of Mouse Thoracic DRG Neurons
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Thoracic DRGs were dissociated from mice euthanized by CO 2 inhalation. The DRGs were enzymatically digested with collagenase type II (Worthington, Lakewood, NJ) and DNase (Roche, Basel, Switzerland) and subsequently with trypsin (Worthington). After trituration, dissociated cells were plated on poly-L-lysine (Sigma-Aldrich, St Louis, MO) coated coverslips and cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with fetal bovine serum (Moregate Biotech, Blimba, Australia or Thermo Fisher, Waltham, MA), and penicillin/streptomycin (Thermo Fisher). For the patch clamp experiments, the coverslips were coated with poly-L-lysine and laminin 511-E8 (iMatrix-511, Nippi, Tokyo, Japan). The cells were used 16-32 h after plating.
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