NPCs were generated as previously described with appropriated optimization27 (link). Briefly, iPSCs were dissociated in cell clusters using Accutase solution (Sigma-Aldrich) and seeded onto low-adhesion plates in mTeSR1 supplemented with 0.5× N-2 supplement (ThermoFisher Scientific), Pen/Strept (1%, Sigma-Aldrich), human Noggin (0.5 µg/ml, PeproTech), SB-431542 (5 µM, Sigma-Aldrich) and ROCK inhibitor Y27632 (10 µM). The medium was replaced every 3 days. After 10 days, embryoid bodies were seeded onto matrigel-coated plates (1:100, Matrigel growth factor reduced, Corning) in DMEM/F12 (Sigma-Aldrich) supplemented with 1× N-2 supplement, nonessential amino acids (1%, MEM NEAA, ThermoFisher Scientific) and Pen/Strept. The medium was replaced every other day. After 10 days, rosettes were dissociated with Accutase solution and NPCs (P0) were plated onto matrigel-coated-flasks in NPC media containing DMEM/F12, 0.5× N-2, 0.5× B-27 supplement (ThermoFisher Scientific), Pen/Strept (1%), and bFGF (20 ng/ml, ThermoFisher Scientific). NPCs were passaged twice a week using Accutase solution; experiments were performed with NPCs between P3 and P10.
Pen strept
Manufactured by Merck Group
Sourced in United Kingdom
Pen/Strept is a laboratory equipment product used for the detection and identification of penicillin and streptomycin in samples. It provides a simple and reliable method for the qualitative analysis of these antibiotics.
Automatically generated - may contain errors
Lab products found in correlation
Mem neaa, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Accutase solution, by Merck Group (3 mentions)
Matrigel, by Corning (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Pen strept, by Thermo Fisher Scientific (2 mentions)
Accutase solution, by Thermo Fisher Scientific (2 mentions)
Sb431542, by Merck Group (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Merck Group (2 mentions)
Glutamine, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Human noggin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate solution, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium high glucose, by Merck Group (1 mentions)
Human noggin, by R&D Systems (1 mentions)
Mifepristone, by Merck Group (1 mentions)
8 protocols using pen strept
1
Neural Progenitor Cell Derivation from iPSCs
2
Fibroblast Culture Protocol for SETBP1 Mutation Analysis
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The patient fibroblasts harboring heterozygous mutations I871T and D868N in the SETBP1 gene and aged-matched control fibroblasts were obtained from Alexander Hoischen (Radboud University Medical Center, Nijmegen, The Netherlands). The protocol was approved by the Medical Ethics Committee of the Radboud University Medical Center and written consent to participate was obtained for all patients. All human skin fibroblast (HSF) cultures were maintained in DMEM medium (Dulbecco’s Modified Eagle’s Medium—high glucose, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% Pen/Strept, 2 mM glutamine (Sigma-Aldrich), 1% nonessential amino acids (MEM NEAA, ThermoFisher Scientific), 1% sodium pyruvate solution (Sigma-Aldrich). The cultures were kept in a humidified atmosphere of 5% CO2 at 37 °C under atmospheric oxygen conditions.
3
Optimized Derivation of Neural Progenitor Cells from iPSCs
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NPCs were derived using an optimized protocol previously published83 (link). iPSCs were dissociated in cell clusters using Accutase solution (Sigma-Aldrich) and seeded onto low-adhesion plates in mTeSR1 supplemented with 0.5X N2 supplement (ThermoFisher Scientific), Pen/Strept (1%, Sigma-Aldrich), human Noggin (0.5 µg/ml, R&D System), SB431542 (5 µM, Sigma-Aldrich) and ROCK inhibitor Y27632 (10 µM). Medium was replaced every 3 days. After 10 days, embryoid bodies were seeded onto matrigel-coated plates (1:100, Matrigel growth factor reduced, Corning) in DMEM/F12 (SigmaAldrich) supplemented with 1X N2 supplement, non-essential amino acids (1%, MEM NEAA, ThermoFisher Scientific) and 1% Pen/Strept. Medium was replaced every other day. After 10 days, rosettes were dissociated with Accutase solution and NPCs (P0) were plated onto matrigel-coated flasks in NPC media containing DMEM/F12, 0.5X N2, 0.5X B27 supplement (ThermoFisher Scientific), Pen/Strept (1%) and bFGF (20 ng/ml, ThermoFisher Scientific). NPCs were passaged twice a week using Accutase solution; experiments were performed with NPCs between P3 and P10.
4
Osteogenic Differentiation with HDAC Inhibitors
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For osteogenic differentiation, when cells at the third passage of culture reached 60–70% confluency, they were induced using osteoinduction medium, composed of DMEM supplemented with 10% FBS, 1% Pen-Strept, 50 μg/mL ʟ-ascorbic acid (Sigma, Gillingham, Dorset, UK), 10 mM glycerol phosphate disodium salt (β-glycerophosphate), and 10 nM dexamethasone (Sigma, Gillingham, Dorset, UK). Cells maintained in the basal culture medium served as the controls. The osteogenic medium was changed twice a week.
Valproic acid sodium salt, MS-275, TSA, SAHA (HDAC inhibitors) and RU-486 (Mifepristone, GR antagonist) were purchased from Sigma.
Cells were treated with 1.5 mM of VPA, 2.5 µM of MS275, 100 nM of TSA, and 1 µM of SAHA for 48 hours.
Valproic acid sodium salt, MS-275, TSA, SAHA (HDAC inhibitors) and RU-486 (Mifepristone, GR antagonist) were purchased from Sigma.
Cells were treated with 1.5 mM of VPA, 2.5 µM of MS275, 100 nM of TSA, and 1 µM of SAHA for 48 hours.
5
Differentiation of iPSCs into Mature Cells
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iPSCs at 70–80% confluence were detached by Accutase solution incubation at 37 °C for 10 min in order to obtain a single-cell suspension. Cells were centrifuged, counted, and seeded onto HESC-qualified Matrigel-coated glass coverslips at a density of 26,000 cells/cm2 in mTeSR1 medium supplemented with 10 µM ROCK inhibitor Y27632 (Selleckchem) and 1% Pen/Strept. Twenty-four hours after seeding, medium was replaced with DMEM/F12 containing 1% Pen/Strept, 2 mM glutamine (Sigma-Aldrich), 1% nonessential amino acids (MEM NEAA, ThermoFisher Scientific), 10% fetal bovine serum (FBS, Sigma-Aldrich). Cells were cultured for 4 weeks with medium replacement three times per week and then analyzed by immunofluorescence.
6
Generating Induced Pluripotent Stem Cells from Human Skin Fibroblasts
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Human skin fibroblasts (HSFs) were reprogrammed to pluripotency using CytoTune®-iPS Reprogramming Kit (Invitrogen) following the manufacturer’s instructions. iPSC cell lines were maintained in feeder-free conditions in mTeSR1 (Stem Cell Technologies) supplemented with Pen/Strept (1%, Sigma-Aldrich) and seeded on human embryonic stem cell (HESC)-qualified Matrigel (Corning)-coated six-well plates; cells were fed daily and passaged in cell clumps weekly using Accutase solution (Sigma-Aldrich). iPSCs were used at passages between 25 and 50 for all the subsequent experiments.
7
Feeder-Free Culture of hiPSC Lines
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SGS iPSCs were the same used in our previous work39 , they are deposited on hPSCreg (https://hpscreg.eu ) which released us certificates (#OSRi009-A, OSRi009-A-1, OSRi010-A, OSRi010-A-1). All iPSCs lines were cultured in feeder-free conditions in mTeSR1 (Stem Cell Technologies) supplemented with Pen/Strept (1%, Sigma-Aldrich) and seeded in six-well plates coated with human embryonic stem cell (HESC)-qualified Matrigel (Corning).
8
Fibroblast Isolation from Fetal Thymic Explants
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Thymic explants were derived from foetal biopsies at different thymic stages (HDBR Newcastle University -NewCcstle Upon Tyne, REC reference: 19/NE/0290 and HDBR University College of London -London, REC reference: 18/LO/0822) and cultured on a precoated Matrigel (Corning) 6mm dish in DMEM (Life Technologies) supplemented with 15% FBS HI (Life Tehnologies) + 1% Pen/Strept (Sigma-Aldrich), 1% L-glutamine (Life Technologies), 1% Non-Essential Aminoacids (Life Technologies) and 100mM beta-Mercaptoethanol (Life Technologies). Fibroblast cells come out of explants at around 7 days of culture and are left on the plate until outgrowths are confluent enough to pass. The culture is therefore kept for 5-6 passages and phenotypic analysis was performed at multiple passages. Fibroblasts were detached with trypsin 1X (Sigma-Aldrich) for 3' at 37C and subsequently resuspended in completed media before collection. Cells are harvested and phenotypic analysis is performed on 500,000 cells per sample. Cells were stained at 4°C for 30 min in Hanks Balanced Salt Solution-2% FBS with the following markers: anti-THY1 AF700 1:100 (Biolegend), anti-PDGFRalpha PE 1:100 (Biolegend) and PI-16 (BD) 1:50. Cells are washed in an excess of HBSS + 2%FBS and are resuspended in HBSS + 2%FBS with DAPI (Sigma-Aldrich) to discriminate live from dead cells.
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