Esgro medium

Manufactured by Merck Group

Sourced in Germany

ESGRO medium is a cell culture medium designed to support the growth and maintenance of embryonic stem cells. It provides the necessary nutrients and growth factors required for the in vitro culture of embryonic stem cells.

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ESGRO (128 citations) ESGRO Complete PLUS Clonal Grade Medium (16 citations) ESGRO-2i medium (14 citations) ESGRO Complete Basal medium (8 citations) ESGRO mouse LIF medium supplement (4 citations) ESGRO Complete Plus Serum-free Clonal Grade 1i Medium (2 citations) ESGRO mLIF medium supplement (2 citations) ESGRO mouse LIF Medium Supplement (Leukemia Inhibitory Factor) (2 citations) ESGRO Complete Plus Serum-free Clonal Grade Medium (2 citations)

5 protocols using esgro medium

Reprogramming of Somatic Cells to Pluripotency

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Mouse ES cells (B6 cell line) were cultured in ESGRO medium (Millipore, Germany) containing leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). The medium was supplemented with glycogen synthase kinase 3β inhibitor (GSK3βi) supplement, which is necessary for maintaining pluripotency of ES cells.¹⁸ (link)
For somatic cells, we used mouse embryonic fibroblast MEFs containing an endogenous Oct4-GFP reporter that fluoresces green, when reprogramming to pluripotency is successfully initiated after fusion. MEFs were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).
Fused cells were cultured in ESGRO medium to avoid differentiation of ES nuclei. However, because ESGRO has low nutrients, it was supplemented with 1% FBS to support the survival of MEFs. GSK3βI was not added to the medium.

Sakamoto S., Okeyo K.O., Yamazaki S., Kurosawa O., Oana H., Kotera H, & Washizu M. (2016). Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids. Biomicrofluidics, 10(5), 054122.

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Retinoic Acid-Induced Differentiation of Mouse Embryonic Stem Cells

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E14 mESC line was from ATCC, and WT and SIRT1 KO mESCs have been described (McBurney et al., 2003 (link)). All stem cells were maintained in the ESGRO Complete Clonal Grade Medium (ESGRO medium, Millipore). To induce the differentiation of mESCs with RA, mESCs were first cultured on gelatin-coated plates in the M10 medium. Sh-Control and sh-SIRT1 mESCs were treated with ethanol or 20 nM RA in the M10 medium for indicated times. WT and SIRT1 KO mESCs were treated with 0.2 μM RA.

Tang S., Huang G., Fan W., Chen Y., Ward J.M., Xu X., Xu Q., Kang A., McBurney M.W., Fargo D.C., Hu G., Baumgart-Vogt E., Zhao Y, & Li X. (2014). SIRT1-mediated deacetylation of CRABPII regulates cellular retinoic acid signaling and modulates embryonic stem cell differentiation. Molecular cell, 55(6), 843-855.

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Efficient Gene Targeting in Mouse ESCs

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E14 and J1 ESCs were obtained from the American Type Culture Collection. Dnmt-TKO and Rosa26-CreERT2 ESCs were kindly provided by Dr Masaki Okano and Dr Shaun Cowley. For experiments, ESCs were routinely cultured on gelatin-coated plates in Dulbecco’s modified Eagle’s medium supplemented with 15% ESC-qualified fetal bovine serum, 10 mM 2-mercaptoethanol, 1 mM non-essential amino acids and 1000 U/ml of LIF (Millipore). For cell line maintenance, ESCs were cultured in the serum-free ESGRO medium (Millipore). ESC differentiation and transfections were carried out similarly as previously described (41 (link)).
For gene targeting, pX330 and homologous recombination (HR) donor plasmids were co-transfected into E14 (for HA-tag knock-in) or Rosa26 CreERT2 (for conditional deletion) ESCs. Transfected cells were seeded at colonal density, and individual colonies were picked and screened by polymerase chain reaction (PCR). Correctly targeted clones were amplified and re-screened to confirm genotype.

Li P., Wang L., Bennett B.D., Wang J., Li J., Qin Y., Takaku M., Wade P.A., Wong J, & Hu G. (2017). Rif1 promotes a repressive chromatin state to safeguard against endogenous retrovirus activation. Nucleic Acids Research, 45(22), 12723-12738.

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Culturing Mouse Embryonic Stem Cells

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E14TG2a mouse ESCs (ATCC) were routinely cultured on gelatin-coated plates in DMEM (GIBCO) supplemented with 15% ESC-qualified fetal bovine serum (GIBCO), 10 mM 2-mercaptoethanol, 1mM nonessential amino acids, and 1,000 U/ml of LIF (Millipore). For cell maintenance, ESCs were cultured in the serum-free ESGRO medium (Millipore). 293T cells were maintained in DMEM with 10% fetal bovine serum (Biological Industries) at 37°C with 5% CO2.

Kim H.J., Li P., Kim T., Oldfield A.J., Zheng X, & Yang P. (2022). Integrative analysis reveals histone demethylase LSD1 promotes RNA polymerase II pausing. iScience, 25(10), 105049.

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Maintenance and Differentiation of Mouse ESCs

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For routine culture, mouse ESCs were maintained in the ESGRO medium (Millipore) on tissue culture plates pretreated with 0.1% gelatin. For the naïve pluripotent state, mouse ESCs were cultured in 2iL medium containing N2B27 medium supplemented with PD0325901 (1 mM, Selleck), CHIR99021 (3 mM, Selleck) and 1000 units/ml recombinant murine LIF (Millipore) on gelatin-coated plates (2iL). For the primed pluripotent state, mouse ESCs were cultured in FA or FAX medium containing N2B27 supplemented with FGF2 (Peprotech, 12 ng/ml), Activin A (Peprotech, 20 ng/mL), and XAV-939 (2 uM Selleck) on human fibronectin-coated plates (Millipore). The N2B27 medium contains (1000 ml): 500 ml DMEM/F12 (Invitrogen; 11320), 500 ml Neurobasal (Invitrogen; 21103), 5 ml N2 supplement (Invitrogen; 17502048), 10 ml B27 supplement (Invitrogen; 17504044) and 0.1% bovine serum albumin fraction V (Thermofisher).

Yu H., Wang J., Lackford B., Bennett B.D., Li J., & Hu G. (2020). INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells.

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