Rw4 murine (male,129X1/SvJ) embryonic stem cells (mESCs) were cultured feeder-free in 0.1% gelatin-coated dishes. Serum condition: Knockout DMEM (Life Technologies), 2 mM Glutamax (GIBCO), 0.1 mM non-essential amino acids (GIBCO), 15% ESC-grade fetal bovine serum (FBS) (GIBCO), 0.1 mM β-mercaptoethanol, and leukemia inhibitory factor (LIF) (Millipore), 2i/Serum condition: above medium supplemented with 2i; 1 μM MEK inhibitor PD0325901 (Sigma) and 3 μM GSK3 inhibitor CHIR99021 (Sigma). 2i condition: serum free ESGRO Complete Basal medium (Millipore) with 0.1mM LIF and 2i as described above. For drug treatment, mESCs were grown in respective medium supplemented with 10 μM GSK-J4 (Sigma) for 96 h, 50 μM Zebularin for 96 h or 10 μM EPZ-6438 (BioVision) for 7 days. For all conditions cells were passaged in 48 h intervals, using accutase (Sigma) for detachment. Cell line was tested for mycoplasma contamination.
Esgro complete basal medium
Manufactured by Merck Group
Sourced in Germany
ESGRO Complete Basal medium is a cell culture medium developed by Merck Group. It is designed to support the growth and maintenance of embryonic stem cells and other pluripotent cell types. The medium provides the necessary nutrients and supplements to ensure optimal cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Accutase, by Merck Group (3 mentions)
Gelatin solution, by Merck Group (2 mentions)
Esgro complete plus clonal grade medium, by Merck Group (2 mentions)
6 cm bacteriological petri dishes, by BD (2 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Leukemia inhibitory factor, by Merck Group (1 mentions)
Mek inhibitor pd0325901, by Merck Group (1 mentions)
Gsk3 inhibitor chir99021, by Merck Group (1 mentions)
Gsk j4, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Mek inhibitor pd0325901, by Fujifilm (1 mentions)
Gsk3 inhibitor chir99021, by Fujifilm (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Roche (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Doxycycline dox, by Takara Bio (1 mentions)
8 protocols using esgro complete basal medium
1
Epigenetic Regulation in Mouse Embryonic Stem Cells
2
Generating Reporter ES Cells
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Female mT/mG mice were superovulated by intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at an interval of 48 h. hCG-injected female mice were mated with Oct4dPE-CreERT2 male mice, and the copulatory plug was confirmed the next morning. Blastocysts were collected from the uterus 3 days later. Collected blastocysts were plated on mitomycin-treated mouse embryonic fibroblast feeder cells in 2i-LIF medium (ESGRO Complete Basal medium; Millipore, Germany) containing leukemia inhibitory factor (LIF) (Wako, Tokyo, Japan), 0.4 µM MEK inhibitor PD0325901 (Wako, Tokyo, Japan), 3 µM GSK3 inhibitor CHIR99021 (Wako, Tokyo, Japan) and Penicillin-Streptomycin (Invitrogen). Outgrowths from blastocysts were disaggregated and passaged to new culture wells containing feeder cells in 2i-LIF medium. Once ES cell colonies emerged, these cells were expanded for genotyping and frozen. ES cells were treated with Proteinase K (Roche) and genotyped using the following primers: [WT Rosa locus; Rosa-F (5′-ctc tgc tgc ctc ctg gct tct-3′) and Rosa-R (5′-cga ggc gga tca caa gca ata-3′), mT/mG locus; Rosa-F and mT/mG R (5′-tca atg ggc ggg ggt cgt t-3′), and CreERT2; CreERT2-F (5′-gaa gca act cat cga ttg att tac gg-3′) and CreERT2-R(5′-tga agg gtc tgg tag gat cat act c-3′)]. We named the established ES cells TGOC referring to Rosa-mT mG /O ct-dPE-C reERT.
3
Expansion of Embryonic Stem Cells
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After feeder depletion, 7 x 104 ESCs were plated in the well of a 24-well plate pre-coated with gelatin. Cells were incubated with ESGRO Complete Basal medium (Millipore, Germany).
4
Derivation and Maintenance of Dis3 Mutant mESC Lines
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Dis3flox/flox female mice10 (link) were stimulated sequentially with PMCG and hCG and mated to Dis3flox/flox male mice. Embryos were flushed from the uterus 3.5 days post coitus with pre-warmed ESGRO Complete Basal Medium (Sigma SF002). All wells of a 24-well plate were treated with 0.5 mL Gelatin solution (Sigma-Aldrich SF008) for 30 min at room temperature. After removing Gelatin, 0.5 mL pre-warmed ESGRO Complete Plus Clonal Grade Medium (Sigma SF001) was added into each well of the plate. Embryos at the blastocyst stage were transferred individually into each well. Embryos were cultured in ESGRO Complete Plus Clonal Grade Medium for 5 days at 37◦C with 5% CO2 and outgrowth of cells was observed from some embryos. Cells were treated with Accutase (Sigma-Aldrich SF006) at 37◦C for 1 min to dissociate into single cells. 1–5 single cells were transferred into fresh ESGRO Complete Plus Clonal Grade Medium, cultured, and maintained as a stable cell line. Cells were split when reaching 50% confluence and forming spheroids of 20–50 cells. For differentiation, cells were dissociated, washed with SF002 medium, and cultured in SF002 medium at 37◦C with 5% CO2 for 3 days. Transfection of mESCs is described in Method details below.
5
Derivation and Maintenance of Dis3 Mutant mESC Lines
6
Embryoid Body Formation for Stem Cell Differentiation
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Induction of differentiation was achieved through embryoid body (EB) formation via hanging drop culture following a procedure adapted from De Smedt et al.[26] (link). In brief, stem cells were thawed and a suspension was prepared at a concentration of 3.75×104 cells/ml in ESGRO Complete Basal Medium (EMD Millipore), which does not contain LIP, BMP-4, or GSK3b-I. About 50 drops (each of 20 µl) of the cell suspension were placed onto the inner side of the lid of a 10-cm Petri dish filled with 5 ml phosphate buffered saline (PBS; EMD Millipore) and incubated at 37°C and 5% CO2 in a humidified atmosphere. After 3 days, EBs formed in the hanging drops (Ø330–350 µm) were subsequently transferred into 6-cm bacteriological Petri dishes (Becton Dickinson Labware, Franklin Lakes, NJ) and were further cultivated for 2 days. On day 5, EBs were plated one per well into 24-well tissue culture plates (Thermo Scientific Nunc, Roskilde, Denmark). During further development of the attached EBs, cells of endodermal, ectodermal and mesodermal origin were obtained in the outgrowths. In EST, differentiation was determined by microscopic inspection of contracting cardiomyocytes in the EB outgrowths on day 10.
7
Stem Cell Differentiation via EB Formation
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Induction of differentiation was achieved through embryoid body (EB) formation in hanging drop culture following a procedure adapted from De Smedt et al. [25 (link)]. In brief, stem cell suspensions were prepared on ice at a concentration of 3.75 × 104 cells/ml in ESGRO Complete Basal Medium (EMD Millipore), which does not contain LIP, BMP-4, or GSK3b-I. About 50 drops (each of 20 µl) of the cell suspension were placed onto the inner side of the lid of a 10-cm Petri dish filled with 5 ml phosphate buffered saline (PBS) (EMD Millipore) and incubated at 37 °C and 5% CO2 in a humidified atmosphere. After 3 days, EBs formed in the hanging drops were subsequently transferred into 6-cm bacteriological Petri dishes (Becton–Dickinson Labware, Franklin Lakes, NJ) and were exposed to AgNPs or Ag+. The EBs had an average diameter of 330–350 μm.
8
Generating M. minutoides Induced Pluripotent Stem Cells
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Fibroblasts from M. minutoides were dissociated, washed, and suspended in Opti-MEM (Gibco). The cell suspension was then transferred to a 4-mm cuvette electrode (SE-204; BEX Co., LTD), and 2.5 μg/100 μL of plasmid was added individually for iPSC induction (PB-TRE-OKSM, PB-TRE-GNL, PB2-CAG-rtTA-ires-eGFP pGK-Neo, and pCAG-T3-hyPBase-pA(95)). Electroporation was performed using a CUY21EDIT II electroporator (BEX) with a single 10 ms pulse at 350 V, followed by five 50 ms pulses at 40 V at 50 ms intervals. After electroporation, the cells were reseeded on feeder cells in ESGRO Complete Basal Medium (Merck) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 20% KnockOut Serum Replacement (KSR) (Gibco), and ESGRO-2i Supplement Kit (Merck), with 2 μg/mL (final concentration) doxycycline (DOX) (Takara). Emerging colonies with iPSC-like morphology were passaged on feeder cells and mitomycin C-treated mouse embryonic fibroblasts and cultured with DOX until the reappearance of the colonies. Subsequently, DOX supplementation was discontinued, and the cells were further cultured. Individual colonies that maintained domed-morphology were selected under a stereomicroscope and single cells from a colony were seeded on feeder cells in 96-well plates to establish multiple M. minutoides iPSC lines.
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