Naive PSCs were maintained in t2iL + PKCi media as previously described22 (link) in a 1:1 mixture of DMEM/F12 and Neurobasal, 0.5× N2 supplement, 0.5× B27 supplement, 1× nonessential amino acids, 2mM l -Glutamine, 1× Penicillin/Streptomycin (all from ThermoFisher Scientific), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1 μM PD0325901, 1 μM CHIR99021, 20 ng/ml human LIF (all from WT-MRC Cambridge Stem Cell Institute) and 2 μM Gö6983 (PKCi; Tocris) on Matrigel-coated plates (Corning). Primed PSCs were maintained on Vitronectin-coated plates (0.5 μg/cm2; ThermoFisher Scientific) in TeSR-E8 medium (StemCell Technologies).
Vitronectin coated plates
Manufactured by Thermo Fisher Scientific
Sourced in United States
Vitronectin-coated plates are a type of cell culture plate designed for the attachment and growth of cells that exhibit a preference for the extracellular matrix protein vitronectin. These plates provide a standardized and optimized surface for cellular adhesion and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Tesr e8 medium, by STEMCELL (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
E8 medium, by Thermo Fisher Scientific (2 mentions)
Essential 8, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium high glucose, by Cytiva (2 mentions)
Ultrapure edta, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Primocin, by InvivoGen (2 mentions)
Y 27632, by STEMCELL (2 mentions)
Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (2 mentions)
Nutristem hpsc xf medium, by Sartorius (2 mentions)
Matrigel coated plate, by Corning (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Pcxle hoct3 4 shp53, by Addgene (1 mentions)
Minimum essential media, by Thermo Fisher Scientific (1 mentions)
Pcxle hsk, by Addgene (1 mentions)
Pcxle hul, by Addgene (1 mentions)
Epi5 episomal ipsc reprogramming kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using vitronectin coated plates
1
Maintaining Naive and Primed Pluripotent Stem Cells
2
Fibroblast Reprogramming to iPSCs
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Fibroblasts were isolated from skin biopsy explants from the proband harboring a biallelic ACTL6B p.(Val421_Cys425del) variant and a sex‐matched unaffected familial carrier (biological mother of the affected patients) described in Yüksel et al. (2019 ). Fibroblasts were cultured in Minimum Essential Media (Gibco), supplemented with 20% (v/v) fetal bovine serum (Gemini) and 1% (v/v) antibiotics (Pen/Strep 10,000 U/ml). Low passage fibroblasts were reprogrammed to iPSCs using Epi5 Episomal iPSC Reprogramming Kit (ThermoFisher) with the following modifications: fibroblasts were electroporated and plated onto Matrigel (Corning)‐coated plates. Reprogramming vectors pCXLE‐hOCT3/4‐shp53 (Addgene, 27077, RRID:Addgene_27077), pCXLE‐hSK (Addgene, 27078, RRID: Addgene_27078), pCXLE‐hUL (Addgene, 27080, RRID:Addgene_27080), and pCXWB‐EBNA1 (Addgene, 37624, RRID:Addgene_37624) were gifts from Shinya Yamanaka and prepared in‐house (Okita et al., 2013 ). The medium was changed to StemFlex (ThermoFisher) on day 15. iPSC colonies were manually collected and individual clones were expanded in feeder‐free conditions on vitronectin‐coated plates (ThermoFisher) for karyotyping (Karyostat; Invitrogen) and characterization of pluripotency.
3
Culturing HEK293T and hiPSC cells
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Human embryonic kidney (HEK293T, American Type Culture Collection, Manassas, VA, USA) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 7.5% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37 °C in a 5% CO2 atmosphere. The human induced pluripotent stem cells (hiPSCs) used in this study were generated from umbilical cord blood mononuclear cells (CBMC)70 (link). hiPSCs were maintained in vitronectin-coated plates (Thermo Fisher Scientific, Waltham, MA, USA), and media were replaced daily with Essential 8 medium (E8, Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37 °C in a 10% CO2 atmosphere.
4
Generation of Induced Neuronal Cells
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The G3 human iPSC line used in this study was a gift from Michael Ward (NIH, Bethesda, MD) and was generated as described in Fernandopulle et al. 2018. The cell line harbors a doxycycline inducible NGN2 constructed located at the safe-harbour AAVS1 locus. The B1 human iPSC line was a gift from Mark Kotter (University of Cambridge, UK) and were generated as described in Pawlowski et al. 2017. This line contains the inducible promotor and NGN2 gene in the AAVS1 locus, with the rtTA repressive element located at the ROSA26 locus. All iPSCs were maintained in TeSR-E8 medium (Stem Cell Technologies) and cultured on Vitronectin coated plates (Thermo Fisher Scientific). Cells were passaged as required with 0.5 mM EDTA (Thermo Fisher Scientific) when reaching 80% confluency.
5
iPSC Generation from Human PBMCs
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Peripheral blood samples were obtained from healthy human donors after obtaining informed consent, with approval of the Institutional Review Board of Seoul St. Mary’s Hospital (KIRB-00613_6-001). Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation using Ficoll-Paque PLUS media (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Isolated PBMCs were cultured in StemSpan ACF (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with StemSpan CC110 for 4 days. Mononuclear cells were then transferred to a 24-well vitronectin-coated plate (BD BioCoat, BD Biosciences, Franklin Lakes, NJ, USA), and Sendai virus (SeV) (CytoTune-iPSC 2.0 Reprogramming kit, Thermo Fisher Scientific, Waltham, MA, USA) was added to give a multiplicity of infection of three (MOI:3). The medium was changed daily until iPSC colonies formed. After manual picking, iPSC lines were maintained on vitronectin-coated plates (Thermo Fisher Scientific) in a TeSR-E8 medium (Stem Cell Technologies). Emerging iPSC colonies were picked individually and expanded for characterization on day 12 after transduction. Cells were cultured in a 37°C incubator with 5% CO2 from days 3 to 21 after transduction.
6
Retinoic Acid Induced hESC Differentiation
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Undifferentiated H9 hESCs were maintained on Vitronectin-coated plates (ThermoFisher Scientific) in TeSR-E8 media (StemCell Technologies). To induce differentiation, hESCs were grown in N2B27 media supplemented with 3 µM retinoic acid for 72 hours. N2B27 media consists of a 1:1 mixture of DMEM/F12 and Neurobasal, 0.5x N2 supplement, 0.5x B27 supplement, 1x nonessential amino acids, 2mM L-Glutamine, 1x Penicillin/Streptomycin (all from ThermoFisher Scientific) and 0.1 mM βmercaptoethanol. All hESCs were cultured in 5% O2, 5% CO2 at 37°C.
7
Palatal Periosteum Tissue Protocol
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Full-thickness palatal periosteum (PP) tissue for immunohistochemistry was fixed in 10% neutral buffered formalin for 24 h at 4°C before being embedded in paraffin. Tissue for isolation of PD-MSCs was collected from the two-thirds of the PP closest to the bone. The top third containing the palatal epithelium was removed and discarded, and the accuracy of this process was validated with histological techniques. PP tissue was minced and grown as explant cultures in serum-and xeno-free E8 medium on vitronectin-coated plates (Thermo Fisher Scientific, Waltham, MA, USA).
8
Maintaining Human Stem Cells and Neuroblastoma Cells
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The human induced pluripotent stem cell lines (NC3-1, passage 13; ihtc-03, passage 16) were presented by Dr. Yan Liu's laboratory at Nanjing Medical University, China. All stem cell lines were maintained on vitronectin-coated plates (Life Technologies, USA) with Essential eight medium (Life Technologies), and changed daily at 37 °C in 5% CO2. Cells were passaged every 5 days through ethylenediaminetetraacetic acid digestion (Lonza, USA).
The human neuroblastoma SH-SY5Y cell line at passage 4 was a gift from Dr. Jun Gao's laboratory (Nanjing Medical University). SH-SY5Y cells were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% (v/v) fetal calf serum (Gibco) at 37 °C in 5% CO2.
The human neuroblastoma SH-SY5Y cell line at passage 4 was a gift from Dr. Jun Gao's laboratory (Nanjing Medical University). SH-SY5Y cells were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% (v/v) fetal calf serum (Gibco) at 37 °C in 5% CO2.
9
Directed Differentiation of hPSCs into Neural Lineages
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hESCs (H9, Passage 40–60, WiCell Agreement NO.16-W0060, mycoplasma contamination testing see Supplementary T4) and iPSCs (ihtc, Passage 10–20, established in our laboratory) were maintained on vitronectin-coated plates (Life Technologies) with Essential eight medium, which was changed daily. Cells were passaged every 5 days through ethylenediaminetetraacetic acid (EDTA) (Lonza) digestion. For neural differentiation, hPSCs were detached by dispase (Life Technologies) to form embryoid bodies (EBs) and then cultured in neural induction medium (NIM) as previously described (Yuan et al., 2015 (link)). After floating for 7 days, EBs were attached on vitronectin-coated surfaces. Rosette structures could be observed at day 10–16. At day 16, rosette clones were detached manually with a 1 ml pipette. Non-neuroepithelial clones were removed at this stage. Neurospheres were continuously floated in NIM and then dissociated by TrypLE (Life Technologies) and plated on vitronectin (Life Technologies) and poly-l-ornithine (Sigma) pre-coated coverslips for further neuronal differentiation. For dorsal differentiation, no morphogen was added. For ventral differentiation, 500 nM SAG was added from day 10 to day 25. In all in vitro experiments examining the induction of LHX6 overexpression, dox was added at 3 μg/mL from day 10 to day 25 for continuous treatment.
10
Embryonic Stem Cell Culture Protocol
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All experiments were approved by the Tri-SCI Embryonic Stem Cell Research Oversight Committee (ESCRO). Human embryonic stem cells (WA01, H1) were cultured in E8 medium (Life technologies, #A1517001) on vitronectin-coated plates (Life technologies, #A14700). HEK293 cells were cultured in DME-HG with 10% fetal bovine serum. hES-MP cells were generated and maintained following the manufacturer’s instructions (STEMCELL Technologies, #05240).
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