Mouse lif

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Mouse LIF is a recombinant protein that functions as a cytokine. It is involved in the maintenance of embryonic stem cell pluripotency.

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Recombinant mouse LIF (6 citations) ESGRO mouse LIF (4 citations) ESGRO mouse LIF medium supplement (4 citations) ESGRO mouse LIF Medium Supplement (Leukemia Inhibitory Factor) (2 citations) ESGRO recombinant mouse LIF (2 citations)

13 protocols using mouse lif

Maintenance of Mouse Embryonic Stem Cells

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Mouse embryonic stem cells (mESCs) were cultured in mESC completed medium (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (Gibco), 1% Glutamine (Gibco), and 1% penicillin–streptomycin (Gibco), 1% MEM non-essential amino acids (Gibco), 1% sodium pyruvate (Gibco), 2% HEPES(Gibco), 0.01% mouse LIF (Sigma), 0.5% 2-mercaptoethanol (Sigma)). Medium was changed every 48 h and passaged when cells reach 80% confluency.
Human embryonic kidney 293 (HEK293) cells were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco), 1% Glutamine (Gibco), and 1% penicillin–streptomycin (Gibco). Medium was changed every 48 h and passaged when cells reached 80% confluency.

Zhou X., Sam T.W., Lee A.Y, & Leung D. (2021). Mouse strain-specific polymorphic provirus functions as cis-regulatory element leading to epigenomic and transcriptomic variations. Nature Communications, 12, 6462.

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Culturing N2A and mouse E14 cells

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Wild-type N2A cells and N2A Rosa26^EGFP+ cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplied with 10% fetal bovine serum (FBS) (Gibco) and passaged three times per week. The mouse E14 cells were maintained in 2i medium, comprising DMEM (Gibco, 11965-092) containing 15% FBS (Gibco), 1000 U/ml mouse Lif, 2 mM glutamine (Sigma), 1% penicillin/streptomycin (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol (Sigma), 0.1 mM non-essential amino acids (Gibco), 1 μM PD0325901 and 3 μM CHIR99021. For puromycin selection, EGFP+ cells were sorted, seeded at 10³ cells/well and selected with 2 mg/ml of puromycin for more than 2 weeks (Supplementary Figure S1A).

Pan H., Yu W, & Zhang M. (2019). Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi’s sarcoma-associated herpesvirus ORF52. Bioscience Reports, 39(10), BSR20191914.

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CRISPR-mediated gene editing in mouse cell lines

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Mouse ESCs were cultured in 2i medium, which consists of Dulbecco's modified Eagle's Medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum (FBS), 1,000 U/mL mouse Lif, 2 mM glutamine (Sigma), 1% penicillin/streptomycin (Thermo Fisher Scientific), 0.1 mM non-essential amino acids (Gibco), 1 μM PD0325901 and 3 μM CHIR99021. Mouse Neuro-2a (N2a) and 3T3 cells were cultured in DMEM/F12 (Gibco) containing 10% FBS, 1% penicillin/streptomycin (Solarbio), and 0.1 mM non-essential amino acids (Gibco).
All cells were cultured at 37 °C in a humid atmosphere of 5% CO₂. Transfection of the cells (mES, N2a, and NIH3T3) was performed using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer's instructions. Plasmids containing sgRNA, Cas9 and a donor were transfected into the cells, and 48 h later, transfection-positive cells were sorted by flow cytometry for further analysis.

Zhao X., Nie J., Tang Y., He W., Xiao K., Pang C., Liang X., Lu Y, & Zhang M. (2020). Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration. Frontiers in Veterinary Science, 7, 199.

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Maintaining Mouse Embryonic Stem Cells

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R1 mESCs from A. Nagy (Samuel Lunenfeld Research Institute, Toronto, Canada) were cultured on feeder free plates coated with 0.1% (w/v) gelatin (Sigma) 37C for 10 min. Medium for mESCs contained DMEM (Invitrogen, Paisley, UK), supplemented with 20% ESC-qualified fetal bovine serum, 1mM sodium pyruvate, 1mM non-essential amino acids (NEAA), 1mM penicillin/streptomycin (all from Invitrogen, Carlsbad), 0.1mM 2- b-mercaptoethanol (Sigma Aldrich, St. Louis, MO), with addition of 10ng/mL mouse LIF (EMD Millipore), 1 mM GSK inhibitor (CHIR99021, Selleckchem) and 1 mM MEK inhibitor (PD0325901, Selleckchem). mESCs were passaged every 2–3 days as a single-cell suspension using 0.05% trypsin/EDTA (Life Technologies).

Hussein A.M., Wang Y., Mathieu J., Margaretha L., Song C., Jones D.C., Cavanaugh C., Miklas J.W., Mahen E., Showalter M.R., Ruzzo W.L., Fiehn O., Ware C.B., Blau C.A, & Ruohola-Baker H. (2020). Metabolic Control over mTOR-Dependent Diapause-like State. Developmental cell, 52(2), 236-250.e7.

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CRISPR-Mediated Knock-In in Mouse Embryonic Stem Cells

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A G4 mouse embryonic stem cell (ESC) line was used in this study (George et al., 2007 (link)). ESCs were maintained under serum-free conditions [0.5 × N-2 (Thermo Fisher Scientific, MA, United States), 0.5 × B-27 (Thermo Fisher Scientific, MA, United States), 100 U/ml mouse LIF (Merck, NJ, United States), 3 μM CHIR99021 (Funakoshi, Tokyo, Japan), 1 μM PD0325901 (Stemgent, MA, United States), 1 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), and 1 × penicillin/streptomycin (Invitrogen, MA, United States) in DMEM/F-12 with GlutaMAX (Thermo Fisher Scientific, MA, United States)] in the absence of feeder layers. The donor vector and pX330 Cas9/single guide RNA vector were transfected into ESCs using Lipofectamine 2000 (Thermo Fisher Scientific, MA, United States). Successful knock-in of the reporter cassette was confirmed by PCR amplification. Primer sets for 5′ and 3′ insertion sites were as follows: forward, 5′-TGAAGCCTGAAACCAGCCCC-3′, reverse, 5′-GCTCGAAGCAGTTGCCCCTCA-3′; and forward, 5′-ACGGCAGTTGGGATTCGTGA-3′, reverse, 5′-CATGATGACCAAGTGTCTGAAAGGG-3′, respectively. The expected PCR product size for wild type is 2,132 bp (5′ forward and 3′ reverse); for knock-in are 4,440 bp (5′ forward and 3′ reverse), 1,357 bp (5′ forward and 5′ reverse) and 1,574 bp (3′ forward and 3’ reverse).

Sheng K.Y., Nakano T, & Yamaguchi S. (2022). A region-dependent allele-biased expression of Dopa decarboxylase in mouse brain. Frontiers in Cell and Developmental Biology, 10, 1078927.

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Culture and Differentiation of Progenitor Cells

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VERO (ATCC No. CRL-1586) and A549 (ATCC No. CCL-185) cells were cultured in Minimum Essential Medium (MEM, TFS, Fr) supplemented with 10% fetal bovine serum (FBS, TFS, Fr). Human neural progenitor cells (hNPCs) were prepared and cultured as described [18 (link)]. Equine iPSCs (eiPSCs) were obtained as described [16 (link)] and cultivated feeder-free using a matrix of truncated vitronectin (Vitro-N, Gibco, TFS, Fr) in a medium composed of StemMACS iPS-Brew (Miltenyi Biotech, Germany) supplemented with mouse LIF (1000 U/mL, Merck Millipore). Neural induction and collection, amplification and banking of eNPCs were achieved as described [19 (link)]. Equine NPCs were maintained on poly-ornithin/laminin coated dishes in N2B27-GF medium [DMEM-F12 with GlutaMAX:Neurobasal (1:1) plus N2, B27 without vitamin A and 0.55 mM 2-mercaptoethanol (TFS, Fr) supplemented with EGF, bFGF and BDNF (10–10–20 ng/mL, respectively, PeproTech)]. Neuronal differentiation of eNPCs (Passage 3 to 8) was induced by EGF and bFGF withdrawal 24 h after plating (125 000 cells/cm² in 96-well plates, Greiner Bio-One). Medium was changed three times a week. All cells were maintained at 37 °C, 5% CO₂. TFS, Thermo Fisher Scientific. Fr, France.

Cochet M., Piumi F., Gorna K., Berry N., Gonzalez G., Danckaert A., Aulner N., Blanchet O., Zientara S., Donadeu F.X., Munier-Lehmann H., Richardson J., Benchoua A, & Coulpier M. (2024). An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus. Veterinary Research, 55, 32.

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Derivation and Maintenance of Haploid Mouse Embryonic Stem Cells

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mAG-haESC derivation and culture were conducted as previously described.¹¹ Briefly, all mAG-haESCs were derived in 15% KSR knockout DMEM (Gibco) supplemented with 0.1 mM mercaptoethanol (Merck Millipore), 1 mM L-glutamine (Merck Millipore), nucleosides (100×, Merck Millipore), 1% nonessential amino acid stock (Merck Millipore), penicillin/streptomycin (100×, Merck Millipore), 2i (1 μM PD0325901 and 3 μM CHIR99021), and 1,000 U/mL mouse LIF (Merck Millipore). Haploid cells were sorted after outgrowth cells expanded to one 12-well plate. Then, mAG-haESCs were propagated in 15% FBS ES medium supplemented with 1 mM L-glutamine, 0.1 mM mercaptoethanol, nucleosides (100×), 1% nonessential amino acid stock, penicillin/streptomycin (100×), 2i, and 1,000 U/mL mouse LIF. Periodic enrichment (every 3∼5 weeks, depending on the cell lines) of haploid cells treated with staining buffer (15 μg/mL Hoechst 33342 and 2.5 μM verapamil) was conducted with a BD FACS Aria II cell sorter.

He W., Tang H., Li Y., Wang M., Li Y., Chen J., Gao S, & Han Z. (2024). Overexpression of Let-7a mitigates diploidization in mouse androgenetic haploid embryonic stem cells. iScience, 27(5), 109769.

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Maintaining mESC Pluripotency via Starvation

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mESCs were serum (fetal bovine serum) and glucose starved for 24 h. Cells were cultured only with DMEM (0 g/L D-glucose, Invitrogen) with addition of 10ng/mL mouse LIF (EMD Millipore), 1 mM GSK inhibitor (CHIR99021, Selleckchem) and 1 mM MEK inhibitor (PD0325901, Selleckchem). To reverse starvation-induced effects, starvation media was removed from the culture after 24 h and mESCs were cultured with DMEM (4.5 g/L D-glucose, Invitrogen), supplemented with 20% ESC-qualified fetal bovine serum, 1mM sodium pyruvate, 1mM non-essential amino acids (NEAA), 1mM penicillin/streptomycin (all from Invitrogen), 0.1mM 2- b-mercaptoethanol (Sigma Aldrich), with addition of 10ng/mL mouse LIF (EMD Millipore), 1 mM GSK inhibitor (CHIR99021, Selleckchem) and 1 μM MEK inhibitor (PD0325901, Selleckchem).

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Isolation and Culture of Cardiac Progenitor Cells

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The isolation and culture of rat cardiac progenitor cells (CPCs) is described in [23 (link)]. Mouse embryonic stem cells (mESCs) were cultured in in high glucose DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U/mL mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC). Two days after embryoid bodies (EBs) were initiated by suspending the cells at 10⁷ cells/mL in 10 mL of media, floating EBs were enzymatically dissociated by treatment with Accutase (e-Bioscience) and were further cultured in hydrogel.
The CHO cells with Notch1 responsive YFP expression were cultured in Alpha MEM containing 10% FBS, 1X Penicillin-Sterptomycin, L-Glutamine, Zeocin (400 μg/mL), Blasticidin (10 μg/mL) and Geneticin (600 μg/mL). The rat cardiac endothelial cells were cultured in low glucose DMEM media (GIBCO) supplemented with 10% FBS, 50 μg/mL endothelial cell growth supplement (Sigma E2759) and 1% 100X MEM Non-essential amino acids solution (GIBCO). The primary rat cardiomyocytes were isolated from rat hearts and cultured as described in [24 (link)]. For 3D cell culture studies, the cells were cultured for 48h in the hydrogels (1R, 1RS, 1RJ, 2R, 2RS, 2RJ; 100μL total volume) in Transwell cell culture insets.

Boopathy A.V., Che P.L., Somasuntharam I., Fiore V.F., Cabigas E.B., Ban K., Brown M.E., Narui Y., Barker T.H., Yoon Y.S., Salaita K., García A.J, & Davis M.E. (2014). The modulation of cardiac progenitor cell function by hydrogel-dependent Notch1activation. Biomaterials, 35(28), 8103-8112.

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Differentiating mouse ESCs into Cardiomyocytes

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mESCs (J1) were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% nonessential amino acids solution, 1% l-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin, and 2000 U/mL mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC). To differentiate mESCs into cardiac lineage, an embryoid body (EB) method was employed with some modifications.^{47 (link)} EBs were formed by suspending the cells at 10⁷ cells/mL in 10 mL of differentiation media: alpha-modified Eagle medium (α-MEM; Invitrogen) supplemented with 10% FBS, 1% nonessential amino acids, 1% l-glutamine, 1% β-mercaptoethanol, l-ascorbic acid (50 μg/mL; Sigma), and 1% penicillin/streptomycin. Differentiation medium was changed every day. Four days after the initiation of EB formation, floating EBs were collected by centrifugation and transferred to fibronectin (Sigma)-coated plates to be attached. Then these attached EBs on fibronectin-coated plates were cultured in nonserum culture medium: DMEM/F12 (Invitrogen) supplemented with l-ascorbic acid (50 μg/mL) for further differentiation into CMs. Typically, beating cells appeared on day 7.

Ban K., Park H.J., Kim S., Andukuri A., Cho K.W., Hwang J.W., Cha H.J., Kim S.Y., Kim W.S., Jun H.W, & Yoon Y.S. (2014). Cell Therapy with Embryonic Stem Cell-Derived Cardiomyocytes Encapsulated in Injectable Nanomatrix Gel Enhances Cell Engraftment and Promotes Cardiac Repair. ACS Nano, 8(10), 10815-10825.

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