236 eg 01m

Manufactured by R&D Systems

Sourced in United States

The 236-EG-01M is a laboratory equipment product. It is a compact and versatile device designed for general-purpose laboratory applications. The product's core function is to perform specific tasks required in a research or testing environment. No further details on the intended use or capabilities of the 236-EG-01M can be provided in an unbiased and factual manner.

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12 protocols using 236 eg 01m

Generating Alveolar Epithelial Cells from hPSCs

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Human embryonic stem cell lines hPSC1 (H9, WiCell) were cultured with mTeSR (StemCell Technologies, Inc., 85850) on Vitronectin XF (StemCell Technologies, Inc., 07180)-coated tissue culture plates. Cells were split in a ratio of 1:6 to 1:12 every 4–6 days using Gentle dissociation reagent (StemCell Technologies, Inc., 07174). Pneumocyte differentiation was induced as previously described (Riva et al., 2020 (link); White et al., 2021 (link)). Briefly, cells were collected at 70%–80% confluency and approximately, 2 million cells per 10 cm² were plated on 12-well Vitronectin-coated tissue culture plates in mTeSR. The next day, definitive endoderm differentiation was induced as previously described (Jacob et al., 2019 (link)). Cells were split after 4 days and further induced to differentiate based on an adapted alveolar differentiation protocol (Ghaedi et al., 2013 (link)) in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies, 31980030) supplemented with 10% FBS (Sigma, F4135), 2 mM L-glutamine (Life Technologies 25030081), 0.5 μM all-trans-retinoic acid (Sigma, R2626), 10 ng/mL FGF-10 (R&D Systems, 345-FG-025), 10 ng/mL EGF (R&D Systems, 236-EG-01M), 100 ng/mL Wnt3a (R&D Systems, 5036-WN-010), 10 ng/mL KGF (R&D Systems, 251-KG-050) and 5 ng/mL BMP-4 (R&D Systems, 314-BP-010). Viral infections were performed on day 11 of differentiation.

Escalera A., Gonzalez-Reiche A.S., Aslam S., Mena I., Laporte M., Pearl R.L., Fossati A., Rathnasinghe R., Alshammary H., van de Guchte A., Farrugia K., Qin Y., Bouhaddou M., Kehrer T., Zuliani-Alvarez L., Meekins D.A., Balaraman V., McDowell C., Richt J.A., Bajic G., Sordillo E.M., Dejosez M., Zwaka T.P., Krogan N.J., Simon V., Albrecht R.A., van Bakel H., García-Sastre A, & Aydillo T. (2022). Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission. Cell Host & Microbe, 30(3), 373-387.e7.

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Glioblastoma Tumor Cell Culture Protocol

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Following informed consent, surgical specimens were obtained from the GBM patient who had brain tumor removal surgery at the Samsung Medical Center (Seoul, Korea) in accordance with the appropriate Institutional Review Boards. Genomic and molecular alterations of these tumors were previously reported. Dissociated GBM cells were cultured in neurobasal media with 0.5X, N2 supplement (17502-048, Invitrogen) and 0.5X, B27 supplement (12587-010, Invitrogen), 25 ng/ml, human recombinant basic fibroblast growth factor (bFGF) (233-FB-01M, R&D system) and 25 ng/ml, epidermal growth factor (EGF) (236-EG-01M, R&D system). U87MG cell line was obtained from and authenticated by American Type Culture Collection. Cells were maintained in Minimum Essential Media (MEM) (11095, Gibco) supplemented with 10% fetal bovine serum (FBS) (12483-030, Gibco) and antibiotics, penicillin/streptomycin (15140-122, Gibco).

Kang W., Kim S.H., Cho H.J., Jin J., Lee J., Joo K.M, & Nam D.H. (2015). Talin1 targeting potentiates anti-angiogenic therapy by attenuating invasion and stem-like features of glioblastoma multiforme. Oncotarget, 6(29), 27239-27251.

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In Vitro Expansion of Glioma Stem Cells

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For in vitro expansion, GSCs were cultured in Neurobasal A media (NBA; 10888-022, Gibco, Waltham, MA, USA) supplemented with N2 and B27 (0.5× each; 17502-048 and 12587-010, Gibco), human recombinant basic fibroblast growth factor, and epidermal growth factor (20 ng/mL each; 233-FB-001MG/CF and 236-EG-01M, R&D Systems, Minneapolis, MN, USA), as well as 100× penicillin streptomycin-glutamine (10378-016, Gibco) [7 (link)]. Next, GSCs were cultured at 37 °C in a humidified incubator containing 5% CO₂.

Lee M., Kim Y.S., Lee K., Kang M., Shin H., Oh J.W., Koo H., Kim D., Kim Y., Kong D.S., Nam D.H, & Lee H.W. (2019). Novel Semi-Replicative Retroviral Vector Mediated Double Suicide Gene Transfer Enhances Antitumor Effects in Patient-Derived Glioblastoma Models. Cancers, 11(8), 1090.

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Isolation and Characterization of Glioblastoma Stem-like Cells

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Freshly resected xenografts were dissected and mechanically dissociated according to the Papain Dissociation protocol (Worthington Biochemical Corporation, New Jersey, cat.no.LK003150). Isolated cells from xenografts were maintained as suspension cultures in complete media containing Neurobasal –A medium (12349-015, Invitrogen), supplemented with B27 minus Vitamin A (12587-010, Invitrogen), EGF (20 ng ml⁻¹) (236-EG-01M, R&D systems), FGF (20 ng ml⁻¹) (4114-TC-01M, R&D systems), GlutaMax (35050-038, Invitrogen) and antibiotics (15140-122, Invitrogen). Cells were allowed to recover for 24 h prior their use in downstream experiments. Matched GBM cancer-stem like cells (GSCs, CD133+) and differentiated GBM cells (DGCs, CD133−) populations were isolated by magnetic (MACS) sorting using CD133 microbeads kit (130-100-857, Miltenyi Biotec). The GSCs were maintained in complete media, whilst the DGCs were maintained as monolayer in DMEM (31966-021, Invitrogen), supplemented with 10% FBS (medium (15140-122, Invitrogen) and antibiotics. Both populations were validated functionally by sphere-forming capacity (ELDA) and expression of stem cell markers by immunoblotting for expression of SOX2 and GFAP.

Obara E.A., Aguilar-Morante D., Rasmussen R.D., Frias A., Vitting-Serup K., Lim Y.C., Elbæk K.J., Pedersen H., Vardouli L., Jensen K.E., Skjoth-Rasmussen J., Brennum J., Tuckova L., Strauss R., Dinant C., Bartek J, & Hamerlik P. (2020). SPT6-driven error-free DNA repair safeguards genomic stability of glioblastoma cancer stem-like cells. Nature Communications, 11, 4709.

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Growth Factor Stimulation Assay

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For signalling experiments, serum/growth-factor-deprived cells (18 h) were treated with insulin or growth factors for the indicated time periods. Final concentrations and stocks were as follows: insulin (Thermo Fisher Scientific/Gibco, A11382II), final concentration, 100 nM; stock concentration, 100 μM (dissolved in 0.1 N HCl then diluted in water to final concentration); IGF-1 (Thermo Fisher Scientific, 291-G1-01M), final concentration, 50 ng ml⁻¹; stock, 100 µg ml⁻¹ (in 0.1% (w/v) BSA/PBS); and EGF (R&D Systems, 236-EG-01M), final concentration, 50 ng ml⁻¹; stock, 100 µg ml⁻¹ (in 0.1% (w/v) BSA/PBS). Stocks were prepared under sterile conditions, aliquoted and stored at −80 °C. Initiation of growth factor stimulation and isotopic tracing as described above were typically concurrent unless otherwise described.

Dibble C.C., Barritt S.A., Perry G.E., Lien E.C., Geck R.C., DuBois-Coyne S.E., Bartee D., Zengeya T.T., Cohen E.B., Yuan M., Hopkins B.D., Meier J.L., Clohessy J.G., Asara J.M., Cantley L.C, & Toker A. (2022). PI3K drives the de novo synthesis of coenzyme A from vitamin B5. Nature, 608(7921), 192-198.

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Pneumocyte Differentiation from hPSCs

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Human embryonic stem cell lines hPSC1 (H9, WiCell) were cultured with mTeSR (Stemcell Technologies, 85850) on Vitronectin XF (Stemcell Technologies, 07180)-coated tissue culture plates. Cells were split in a ratio of 1:6 to 1:12 every 4–6 days using Gentle dissociation reagent (Stemcell Technologies, 07174). Pneumocyte differentiation was induced as previously described (Riva et al., 2020 (link); White et al., 2021 (link)). Briefly, cells were collected at 70–80% confluency and approximately, 2 million cells per 10 cm² were plated on 12-well Vitronectin-coated tissue culture plates in mTeSR. The next day, definitive endoderm differentiation was induced as previously described (Jacob et al., 2019 (link)). Cells were split after 4 days and further induced to differentiate based on an adapted alveolar differentiation protocol (Ghaedi et al., 2013 (link)) in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies, 31980030) supplemented with 10% FBS (Sigma, F4135), 2 mM L-glutamine (Life Technologies 25030081), 0.5 μM all-trans-retinoic acid (Sigma, R2626), 10 ng/ml FGF-10 (R&D Systems, 345-FG-025), 10 ng/ml EGF (R&D Systems, 236-EG-01M), 100 ng/ml Wnt3a (R&D Systems, 5036-WN-010), 10 ng/ml KGF (R&D Systems, 251-KG-050) and 5 ng/ml BMP-4 (R&D Systems, 314-BP-010). Viral infections were performed on day 11 of differentiation.

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Cerebellar Progenitor Cell Isolation

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The cerebellum at P0 was dissected in ice-cold HBSS (Gibco) under a microscope. Tissues were then digested with an Accutase (SCR005, Millipore) at 37 °C for 20 min. After the supernatants were removed, the remaining tissues were gently triturated for 10–12 strokes. Cell suspensions were filtered using a 70 μm strainer (BD) and then separated by gradient centrifugation with Percoll solutions (30% and 65%) (GE Healthcare). Purified progenitor cells were collected from the interface and then washed with HBSS. Cells were resuspended, counted and plated in untreated 24-well plates (Corning) at the density of 500,000 cells/ml. Cells were then cultured in Neurobasal Medium (21103-049, Gibco) containing 2% B-27 (17504-044, Gibco), 2 mM GlutaMAX-I (Gibco), EGF (20 ng/ml, 236-EG-01M, R&D), bFGF (20 ng/ml, 4114-TC-01M, R&D) and 1% penicillin/ streptomycin (15140122, Gibco) in a 5% CO₂ incubator at 37 °C. Cells were cultured for 7 days.

Cheng Q., Wu J., Xia Y., Cheng Q., Zhao Y., Zhu P., Zhang W., Zhang S., Zhang L., Yuan Y., Li C., Chen G, & Xue B. (2023). Disruption of protein geranylgeranylation in the cerebellum causes cerebellar hypoplasia and ataxia via blocking granule cell progenitor proliferation. Molecular Brain, 16, 24.

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Cell Migration Tracking in Chemotaxis Assay

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U373 cells were seeded into the narrow (1000 × 2000 × 70 μm) channel of a tissue culture treated μ-slide chemotaxis chamber (Ibidi, 80326). After an overnight incubation, the chemotaxis chamber was filled with DMEM containing 20 ng/ml EGF (R&D Systems, 236-EG-01M). The observation area within the channel was imaged by phase-contrast microscopy via a 10X objective. Images were captured every 10 min for 16 h, and cell migration tracks between 4 and 16 h were analyzed with ImageJ using a manual tracking plugin and with the Chemotaxis and Migration Tool (Ibidi).

Chen J., Ananthanarayanan B., Springer K.S., Wolf K.J., Sheyman S.M., Tran V.D, & Kumar S. (2019). Suppression of LIM Kinase 1 and LIM Kinase 2 Limits Glioblastoma Invasion. Cancer research, 80(1), 69-78.

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Transwell Migration Assay Protocol

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Transwell inserts of 8 and 3 μm pore sizes (Corning, 3422) were functionalized with 100 μg/ml laminin (Invitrogen, 23017–015) for 3 h at 37 °C and then seeded in the upper chamber with 8000 cells per insert in basal medium. The bottom chamber was filled with basal media supplemented with 20 ng/ml EGF (R&D Systems, 236-EG-01M) as a chemoattractant. Cells were allowed to migrate for 24 h and were then fixed with 4% PFA for 15 mins and washed 3X with PBS. Wells were then stained with propidium iodide (Cell Signaling, 4087S) overnight and imaged with a Nikon TE200E2 microscope. Cell counting was analyzed with ImageJ (National Institutes of Health).

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Establishment of Human Trophoblast Stem Cells

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Establishment and culture of blastocyst-derived hTSC lines was performed according to the published method.^{43 (link)} Briefly, blastocyst was placed into Col IV (Corning, 354233)-coated 96-well plate and cultured in 150 µL of hTSC medium. After 4–5 days of culture, the blastocyst outgrowth was dissociated with 50% TrypLE for 10–20 min at 37 °C and passaged to a new Col IV-coated 96-well plate. hTSCs were routinely passaged every 3–4 days at a 1:3 split ratio and pure hTSC lines were established within 5–10 passages. hTSC medium was composed of the following ingredients: DMEM/F12 supplemented with 0.1 mM β-mercaptoethanol, 0.2% FBS (BI, 04-002-1 A), 0.3% BSA, 1% ITS-X supplement (ThermoFisher Scientific, 51500-056), 50 μg/mL L-ascorbic acid 2-phosphate, 50 ng/mL EGF (R&D Systems, 236-EG-01M), 2 μM CHIR99021, 0.5 μM A83-01, 1 μM SB431542, 0.8 mM VPA and 5 μM Y27632. Alternatively, hTSCs can also be cultured on feeders instead of Col IV in hTSC medium.

Ai Z., Niu B., Yin Y., Xiang L., Shi G., Duan K., Wang S., Hu Y., Zhang C., Zhang C., Rong L., Kong R., Chen T., Guo Y., Liu W., Li N., Zhao S., Zhu X., Mai X., Li Y., Wu Z., Zheng Y., Fu J., Ji W, & Li T. (2023). Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Research, 33(9), 661-678.

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