Esgro 2i medium, by Merck Group - Product details

1

Generating Knockout Cell Lines for DNA Methylation Studies

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Rhee et al. previously generated a HCT116 DNMT1 knockout construct in which exons 3, 4, and 5 of human DNMT1 were replaced with a hygromycin resistance gene. The disruption of DNMT1 led to a reduced global DNA methylation^{30 (link)}. Human colon cancer HCT116 wildtype cells (ATCC^® CCL-247^™) and DNMT1^−/− cells were cultured in RPMI medium 1640 (Thermo Fisher Scientific, Waltham, USA) with 10% FBS and 1% P/S.
CCE mESCs (ATCC^® SCRC-1023^™) were cultured in KO-DMEM (Thermo Fisher Scientific), supplemented with 15% fetal bovine serum, 1% Penicillin-Streptomycin, 1% MEM non-essential amino acids, 1% L-Glutamine, 0.2% HEPES, 0.1% 2-Mercaptoethanol (all Thermo Fisher Scientific) and 0.1% Leukemia inhibitory factor (Merck, Darmstadt, Germany). Cells were split every second day. To obtain naïve mESCs, the cells were adapted to a serum-free culture condition using ESGRO^®-2i Medium (Merck) according to the manufacturer’s protocol. The adaption took 14 days. Every second day, cells were split.

Daum R., Brauchle E.M., Berrio D.A., Jurkowski T.P, & Schenke-Layland K. (2019). Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging. Scientific Reports, 9, 7014.

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Feeder-free Culture of Mouse iPSCs

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The miPSCs (male mouse cell line iPS-MEF-Ng-492B-4) was purchased from CiRA, Kyoto University, Japan. This cell line was reprogrammed from mouse embryonic fibroblasts of Nanog-GFP-IRES-Puro r transgenic mice without integration of exogene transfection [45] . The initial culture of miPSCs was performed using mitomycin C treated feeder cells and 0.1% (w/v) gelatin-coated tissue culture dish. The cells were then adapted to xeno-and feeder-free conditions by culturing on 10 µg/ cm 2 of Cultrex mouse laminin I (Bio-Techne GmbH, Germany) coated tissue culture treated plate (TCP) in ESGRO-2i medium (Merck Chemicals GmbH, Germany). The medium was daily changed and the cells from passages 10 to 20 were used for all experiments.

Xu X., Wang W., Li Z., Kratz K., Ma N, & Lendlein A. (2016). Surface geometry of poly(ether imide) boosts mouse pluripotent stem cell spontaneous cardiomyogenesis via modulating the embryoid body formation process. Clinical hemorheology and microcirculation, 64(3).

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3

Metabolic Labeling and Click-iT Analysis of mESCs

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mESCs were cultured in methionine-free, ESGRO 2i medium (Millipore) with antibiotics (1, 0.5, 0.1, and 0.05 μg/mL puromycin or 1, 0.5, 0.1, and 0.05 μg/mL G418) for 4 hrs. Azidohomoalanine was added at 25 μM to the medium during the incubation period [35 (link)]. mESCs were harvested as above, and cell pellets were resuspended in lysis buffer (50 mM Tris, 0.1% SDS with pH adjusted to 8.0). Proteins were labeled with Alexa 488-alkyne and the Click-iT Protein Reaction Buffer Kit according to the manufacturer’s protocol (Thermo Fisher). After reaction, proteins were precipitated with two volumes of ice-cold acetone, and pellets were resuspended in 100 μL PBS containing 8 M urea. Fluorescence was measured in a microplate reader (M1000 Pro, Tecan).

Durmaz Y.T., Shatadal A, & Friend K. (2022). Geneticin reduces mRNA stability. PLoS ONE, 17(7), e0272058.

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4

Cell Culture Protocols of Various Cell Lines

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Human U251, 293T, and PANC1 cells, rhesus monkey FrhK4 cells, African Green Monkey Vero and CV1 cells, Dog D17 and DK cells, mouse Min6 cells, feline CRFK cells, bovine MDBK cells, and rat NRK cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and antibiotics as described previously^{24 (link)}. Mouse iPSCs were generated from murine fibroblasts by lentiviral transduction of four reprogramming factors^{25 (link)} and cultured without feeder cells on Matrigel-coated plates (BD Bioscience) in ESGRO-2i medium (Millipore) supplemented with 45% Dulbecco’s modified Eagle’s medium (DMEM), 5% FCS, 25 U/ml penicillin, and 25 µg/ml streptomycin. Transgene-free iPSCs were reported previously^{26 (link)}, which were cultured in Pluriton medium (Stemgent, Cambridge, MA) supplemented with 25% mTeSR1 medium (Stemcell Technology) and 50 U/ml penicillin and 50 µg/ml streptomycin. Adipose-derived MSCs were derived and cultured as described previously^{27 (link)}.

Ikeda Y., Makino A., Matchett W.E., Holditch S.J., Lu B., Dietz A.B, & Tomonaga K. (2015). A novel intranuclear RNA vector system for long-term stem cell modification. Gene therapy, 23(3), 256-262.

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5

Murine Fibroblast Reprogramming Protocol

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Example 11

Tail-tip fibroblast (TTF) cultures were established from 3-8 day old reprogrammable mice homozygous for both the tet-inducible OSKM polycistronic cassette and the ROSA26-M2rtTA allele (Carey, B. W. et al., 2010, supra). Maintenance of animals and tail tip excision were performed according to a mouse protocol approved by the Caltech Institutional Animal Care and Use Committee (IACUC). TTFs (+doxycycline), iPS cells, and ES cells were cultured in ES medium (DMEM, 15% FBS, sodium bicarbonate, HEPES, nonessential amino acids, penicillin-streptomycin, L-glutamine, b-mercaptoethanol, 1000 U/ml LIF) and grown on 6-well plates coated with 0.1% gelatin and irradiated MEF feeder cells (GlobalStem). For “2i” conditions, iPS cells were grown in ESGRO-2i medium (Millipore). For lncRNA loss-of-function, iPS cells were transfected with siRNAs (IDT) using Lipofectamine RNAiMAX (Life). For SSEA-1 detection, StainAlive SSEA-1 DyLight 488 antibody (Stemgent) was used to detect SSEA-1 positive cells at specified time-points during reprogramming, which were isolated using flow cytometry on an iCyt Mission Technology Reflection Cell Sorter inside a Baker Bioguard III biosafety cabinet.

US09862943B1. Long noncoding RNAs and cell reprogramming and differentiation (2018-01-09). CALIFORNIA INSTITUTE OF TECHNOLOGY [US]. Inventors: Daniel H. Kim [US], Barbara J. Wold [US].

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6

Snail1 Knockout in Mouse ESCs

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Snai1^fl/fl, CAG-Cre + ESC lines were cultured atop feeder layers of mitomycin C-treated mouse embryonic fibroblasts in standard ESC culture medium (DMEM, 15% FBS; 1 × pen/strep; 4 mM glutamine, 50 μM β-mercaptoethanol; 1 × MEM non-essential amino acids, 1 × sodium pyruvate and 1,000 U ml ⁻¹ LIF). Medium changes were performed daily with ESCs passed every 2 days. For serum-free conditions, ESCs were cultured atop feeder layers in ESGRO-2i medium (SF016-100; Millipore). To generate Snail1 KO ESCs, Snai1^fl/fl; CAG-Cre + ESCs were treated with 100 nM 4-OHT for 2 d with untreated cells serving as the isogenic control. Each line of control and 4-OHT-treated ESCs were expanded and frozen for future use. To induce ESC differentiation, stem cells were recovered from feeder layers by trypsinization and seeded successively atop 0.1% gelatin-coated plastic dishes at 37 °C to remove contaminating fibroblasts. Embryoid bodies (EBs) were formed in differentiation media (ESC culture media with 10% FBS in the absence of LIF) in hanging droplets containing 2,000 feeder-free ESCs per 20 μl drop on petri dishes for 2 d. EBs were collected from the drops and cultured in ultralow attachment dishes in differentiation medium (counted as ‘day 0’), with the media being changed every 2 d.

Lin Y., Li X.Y., Willis A.L., Liu C., Chen G, & Weiss S.J. (2014). Snail1-dependent control of embryonic stem cell pluripotency and lineage commitment. Nature communications, 5, 3070.

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7

Culturing Mouse and Human Cell Lines

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Mouse V6.5 ESC (male) and mouse Nanog-Venus ESC (male) lines were grown in ESGRO-2i medium (SF016–200, Millipore) supplemented with 100 units/mL streptomycin and 100 mg/mL penicillin on cell culture dishes coated with 0.1% gelatin (G1890, Sigma-Aldrich). The media was changed every day and cells were passaged every 2 days using Accutase cell detachment solution (SCR005, Millipore). The human HEK293T (female) cells were grown in DMEM/F12 supplemented with 10% Fetal Bovine Serum (FBS), 100 units/mL streptomycin, and 100 mg/mL penicillin. All cell culture experiments were done at 37°C and 5% CO₂. Cells were checked for mycoplasma contamination every three months and were never tested positive.

Shariati S.A., Dominguez A., Xie S., Wernig M., Qi L.S, & Skotheim J.M. (2019). Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9. Molecular cell, 74(3), 622-633.e4.

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8

Murine Fibroblast Reprogramming Protocol

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Example 11

Tail-tip fibroblast (TTF) cultures were established from 3-8 day old reprogrammable mice homozygous for both the tet-inducible OSKM polycistronic cassette and the ROSA26-M2rtTA allele (Carey, B. W. et al., 2010, supra). Maintenance of animals and tail tip excision were performed according to a mouse protocol approved by the Caltech Institutional Animal Care and Use Committee (IACUC). TTFs (+doxycycline), iPS cells, and ES cells were cultured in ES medium (DMEM, 15% FBS, sodium bicarbonate, HEPES, nonessential amino acids, penicillin-streptomycin, L-glutamine, b-mercaptoethanol, 1000 U/ml LIF) and grown on 6-well plates coated with 0.1% gelatin and irradiated MEF feeder cells (GlobalStem). For “2i” conditions, iPS cells were grown in ESGRO-2i medium (Millipore). For lncRNA loss-of-function, iPS cells were transfected with siRNAs (IDT) using Lipofectamine RNAiMAX (Life). For SSEA-1 detection, StainAlive SSEA-1 DyLight 488 antibody (Stemgent) was used to detect SSEA-1 positive cells at specified time-points during reprogramming, which were isolated using flow cytometry on an iCyt Mission Technology Reflection Cell Sorter inside a Baker Bioguard III biosafety cabinet.

US10533177B1. Long noncoding RNAs and cell reprogramming and differentiation (2020-01-14). CALIFORNIA INSTITUTE OF TECHNOLOGY [US]. Inventors: Daniel H. Kim [US], Barbara J. Wold [US].

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9

Cell Culture Protocols of Various Cell Lines

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Human U251, 293T, and PANC1 cells, rhesus monkey FrhK4 cells, African Green Monkey Vero and CV1 cells, Dog D17 and DK cells, mouse Min6 cells, feline CRFK cells, bovine MDBK cells, and rat NRK cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and antibiotics as described previously^{24 (link)}. Mouse iPSCs were generated from murine fibroblasts by lentiviral transduction of four reprogramming factors^{25 (link)} and cultured without feeder cells on Matrigel-coated plates (BD Bioscience) in ESGRO-2i medium (Millipore) supplemented with 45% Dulbecco’s modified Eagle’s medium (DMEM), 5% FCS, 25 U/ml penicillin, and 25 µg/ml streptomycin. Transgene-free iPSCs were reported previously^{26 (link)}, which were cultured in Pluriton medium (Stemgent, Cambridge, MA) supplemented with 25% mTeSR1 medium (Stemcell Technology) and 50 U/ml penicillin and 50 µg/ml streptomycin. Adipose-derived MSCs were derived and cultured as described previously^{27 (link)}.

Ikeda Y., Makino A., Matchett W.E., Holditch S.J., Lu B., Dietz A.B, & Tomonaga K. (2015). A novel intranuclear RNA vector system for long-term stem cell modification. Gene therapy, 23(3), 256-262.

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10

Reprogrammable Mouse Tail-Tip Fibroblasts

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Tail-tip fibroblast (TTF) cultures were established from 3-8 day old reprogrammable mice homozygous for both the tet-inducible OSKM polycistronic cassette and the ROSA26-M2rtTA allele (Carey et al., 2010 (link)). Maintenance of animals and tail tip excision were performed according to a mouse protocol approved by the Caltech Institutional Animal Care and Use Committee (IACUC). TTFs (+ doxycycline), iPS cells, and ES cells were cultured in ES medium (DMEM, 15% FBS, sodium bicarbonate, HEPES, nonessential amino acids, penicillin-streptomycin, L-glutamine, b-mercaptoethanol, 1000 U/ml LIF) and grown on 6-well plates coated with 0.1% gelatin and irradiated MEF feeder cells (GlobalStem). For “2i” conditions, iPS cells were grown in ESGRO-2i medium (Millipore). For lncRNA loss-of-function, iPS cells were transfected with siRNAs (IDT) using Lipofectamine RNAiMAX (Life). For SSEA-1 detection, StainAlive SSEA-1 DyLight 488 antibody (Stemgent) was used to detect SSEA-1 positive cells at specified time-points during reprogramming, which were isolated using flow cytometry on an iCyt Mission Technology Reflection Cell Sorter inside a Baker Bioguard III biosafety cabinet.

Kim D.H., Marinov G.K., Pepke S., Singer Z.S., He P., Williams B., Schroth G.P., Elowitz M.B, & Wold B.J. (2015). Single cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming. Cell stem cell, 16(1), 88-101.

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Esgro 2i medium

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14 protocols using esgro 2i medium

Generating Knockout Cell Lines for DNA Methylation Studies

Feeder-free Culture of Mouse iPSCs

Metabolic Labeling and Click-iT Analysis of mESCs

Cell Culture Protocols of Various Cell Lines

Murine Fibroblast Reprogramming Protocol

Snail1 Knockout in Mouse ESCs

Culturing Mouse and Human Cell Lines

Murine Fibroblast Reprogramming Protocol

Cell Culture Protocols of Various Cell Lines

Reprogrammable Mouse Tail-Tip Fibroblasts

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