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6 protocols using mtesr1

1

Directed Differentiation of hPSCs to Anterior Foregut

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Confluent hPSC cultures were treated with Acutase (STEMCELL Technologies) to resuspend as single cells in mTeSR1 and Y-27632 (10 μM, Tocris) and plated on Matrigel. On the following day, differentiation into definitive endoderm was carried out as previously described (McCracken et al., 2014 (link)). Briefly, cells were treated with Activin A (100 ng mL-1, R&D systems, Minneapolis, MN) and BMP4 (50 ng mL-1, R&D systems) on the first day in RPMI 1640 media (Life Technologies). Cells in the following two days were treated with only Activin A (100 ng mL-1) in RPMI 1640 with increasing concentrations 0.2% and 2% of HyClone defined fetal bovine serum (dFBS, GE Healthcare Life Sciences).
For anterior foregut monolayer cultures, cells were treated for 3 days in Noggin (200 ng mL-1) in RPMI 1640 with 2% dFBS, with all-trans retinoic acid (2 μM, Sigma, St. Louis, MO) the 3rd day.
Alternately, for the generation of anterior foregut spheroids, from definitive endoderm, cells were treated with FGF4 (500 ng mL-1, R&D systems), Noggin (200 ng mL-1) for 3 days in RPMI 1640 with 2% dFBS. Additional factors were tested during this time (described in results), such as CHIR99021 (“chiron” or “chr”, 2 μM, Tocris), Wnt3a (500 ng mL-1, R&D systems), SB431542 (10 μM, Tocris), DEAB (10 μM, Sigma), and retinoic acid (2 μM).
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2

CRISPRi-iPSCs for PD Modeling

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Human CRISPRi-iPSCs (male WTC11 background) were seeded onto hESC-qualified Matrigel (Corning, #354277)–coated cell culture dishes and maintained in mTESR1 (STEMCELL Technologies, # 05850) medium. When 80 to 90% confluent, CRISPRi-iPSCs were dissociated using Versene (Gibco #15040-066) and split at a 1:6 ratio every 4 to 5 days onto Matrigel-coated dishes at the desired density in mTESR1 supplemented with 10 nM Y-27632 dihydrochloride ROCK inhibitor (Tocris #125410). PD patient–derived iPSCs carrying the SNCA triplication (4C) along with the piggyBac Ngn2 cassette and the mutation-corrected isogenic control iPSC lines (2C) with Ngn2 were a gift from V.K. (Brigham and Women’s Hospital), and the generation of these lines is described in detail in the Supplementary Materials.
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3

CRISPR/Cas9 Genome Editing in iPSCs

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Streptococcus pyogenes Cas9 target sites were identified using the online CRISPR design tool (https://crispr.mit.edu) or ChopChop (http://chopchop.cbu.uib.no/). Two to three guides were selected per location and in vitro cutting was assessed using HEK293T cells. Finalised gRNAs were selected based on strength of T7E1 assay (New England Biolabs) and proximity to the desired editing loci. iPSCs were dissociated with Accutase, resuspended in mTesR1 supplemented with Y-27632 (Tocris). Dissociated cells were then immediately transfected with 6 μg of PX459 pSpCas9(BB)-2A-Puro V2.0 (Addgene) and 2 μg of phosphorothioate-treated ssODN (Integrated DNA Technologies) using LT-1 (Mirusbio) reagent. Puromycin (0.3 μg/ml to 0.35 μg/ml) (ThermoFisher) was added to the cells 18 h post-transfection for 48-72 h. Following selection, cells were plated at limiting densities for single clone isolation. Isolated iPSC colonies were manually dissected and picked using a 21G needle; selected colonies were then expanded for DNA analysis. Clones were initially screened using diagnostic restriction digest or via PCR specific primers. Positive clones were subsequently confirmed using Sanger sequencing.
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4

Induction and Maintenance of iPSC

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An overview of all lines is present in Table S4 (Online Resource 17). The CV-B iPSC-line has been described previously [18 (link)]. The study approval for Ctrl-1-SC11 was granted by the local ethics committee (No. 4485, FAU Erlangen-Nuernberg, Germany). IPSC were cultured on Matrigel coated dishes and fed all 24 h with mTesR1 (Stemcell Technologies). Upon reaching ~ 80% confluency, iPSC were passaged using Accutase in a 1:3–1:6 ratio. After passaging and thawing, mTesR1 was supplemented with 10 µM Y27632 (ROCK-inhibitor, RI, Tocris).
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5

Pluripotent Stem Cell Differentiation Assay

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The pluripotent stem cell lines, hESCs (WA09) and hiPSCs (MR90-1) from WiCell Research Institute, WI, USA were used at passages 29 to 33. Cells were cultured in mTeSR1 (STEMCELL Technologies Canada Inc., Vancouver, BC) in Matrigel Matrix (Corning, NY, US) as recommended by the manufacturer. The hESCs and hiPSCs were either treated with 10 μM of Y-27632 ROCK inhibitor or not. Samples were collected at different time-points, 0-untreated, 12, 24, 48, 96 hours (h) and tested over the expression of pluripotency markers (Fig. 1A). The earliest time point that meaningful analysis could be performed is 12 h as the single cells need sufficient time to attach to the culture surface. Samples treated with ROCK inhibitor (Y-27632) where cultured in mTeSR1 with 10 μΜ Y-27632 (dihydrochloride - Tocris Bioscience, Bristol, UK) for 2 hours. Thereafter, cells were detached with the use of Accutase (StemPro - Thermo Fisher Scientific, MA, USA), colonies were dissociated into single cells by pipetting22 (link)23 (link)24 (link), seeded at 30–40 * 104 cells/cm2 density on Matrigel and cultured in mTeSR1 with 10 μΜ Y-27632 up to 96 h. Samples from three independent experiments (N = 3) were collected.
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6

Undifferentiated hESC Maintenance Protocol

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Cell Culture MIXL1-GFP reporter HES3 hESCs (Davis et al., 2008) and H7 hESCs were maintained in an undifferentiated state using mTeSR1 (STEMCELL Technologies) on Geltrex (A1569601; Thermo Fisher Scientific)-coated cell culture plates as previously described (Loh et al., 2016) . hESCs were passaged when they reached $70%-80% confluency by dissociation using Accutase (A1110501; Thermo Fisher Scientific) to obtain a single-cell suspension. For routine maintenance, hESCs were then plated in mTeSR1 supplemented with the ROCK inhibitor Thiazovivin (2 mM; Tocris) for the first 24 hr after plating to enhance cell survival. hESC experiments were conducted under the cognizance of the Stanford Stem Cell Research Oversight (SCRO) Committee.
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