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Rhegf

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RhEGF is a recombinant human Epidermal Growth Factor (EGF) product. EGF is a potent mitogenic factor that stimulates the growth of various cell types, including epithelial and endothelial cells. RhEGF is a purified protein suitable for use in cell culture applications.

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Lab products found in correlation

Heparin, by Merck Group (3 mentions) Rh bfgf, by R&D Systems (3 mentions) N2 supplement, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Poly l ornithine, by Merck Group (2 mentions) Rhfgf basic, by R&D Systems (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Accutase, by STEMCELL (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Dispase 2 protease, by Merck Group (2 mentions) B27 supplement, by Thermo Fisher Scientific (1 mentions) Dmem f12 glutamax medium, by Thermo Fisher Scientific (1 mentions) Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (1 mentions) Laminin, by Merck Group (1 mentions) Ultra low attachment 6 well plate, by Corning (1 mentions) Hydrocortisone, by Merck Group (1 mentions) Gelcount mammalian cell colony counter, by Oxford Optronix (1 mentions) B 27 serum free supplement, by Thermo Fisher Scientific (1 mentions) Insulin, by Merck Group (1 mentions)

26 protocols using rhegf

1

Generating Down Syndrome iPSC-Derived NPCs

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We previously established iPSCs with HSA21 by transducing fibroblast cells from one male and one female (DS1 and DS2) with characteristic DS features and a full T21, as well as from two age and gender-matched healthy individuals (Ctrl1 and Ctrl2) [10 ]. Fibroblasts were reprogrammed using a non-integrating method with CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific, cat no: A16517). Standard karyotype analysis confirmed T21 or euploidy in iPSC derived from DS patients and healthy donors, respectively. Selected iPSC lines were induced to neural progenitor cells (NPC) as described [10 ]. The NPCs were grown on 0.1 mg poly-L-ornithine (Sigma-Aldrich, cat no.: P4957-50ML) and 1 μg/mL laminin (Sigma-Aldrich, cat no: L2020-1MG)-coated plates in DMEM/F12 GlutaMAX medium (Gibco, cat no.: 31331028) supplemented with 10 ng/mL rhFGF-basic (R&D Systems, cat no.: 233-FB-010), 10 ng/mL rhEGF (R&D Systems, cat no.: 236-EG-200), B27 supplement (1:1000, Gibco, cat no.: 08-0085SA), N2 supplement (1:100, Gibco, cat no.: 17502-048), and 1% penicillin/streptomycin (Gibco, cat no.: 15140-122). Self-renewing NPCs derived from the four donors were differentiated for 30 days into DiffNPCs using an undirected protocol (Fig. 1) as described [10 , 24 ].
Laan L., Klar J., Sobol M., Hoeber J., Shahsavani M., Kele M., Fatima A., Zakaria M., Annerén G., Falk A., Schuster J, & Dahl N. (2020). DNA methylation changes in Down syndrome derived neural iPSCs uncover co-dysregulation of ZNF and HOX3 families of transcription factors. Clinical Epigenetics, 12, 9.
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2

Mammosphere Assay for Stem Cell Enrichment

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Mammosphere assay conditions have been previously described31 (link),45 (link). Briefly, single cell suspensions were seeded in 6-well ultra-low attachment plates (Corning) in serum-free DMEM/F12 with 20 ng/ml rhEGF (R&D Systems), hydrocortisone (Sigma) and 1× B27 (Invitrogen). Cell viability was assessed with Thiazolyl Blue Tetrazolium Bromide staining and the number of mammospheres measuring >100 µm was determined in an automated fashion using the GelCount mammalian cell colony counter (Oxford Optronix).
Balko J.M., Schwarz L.J., Cook R.S., Estrada M.V., Giltnane J.M., Sanders M.E., Sánchez V., Dean P.T., Wang K., Combs S.E., Hicks D., Pinto J.A., Landis M.D., Chang J.C., Doimi F.D., Gómez H., Rimm D.L., Yelensky R., Miller V.A., Stephens P.J, & Arteaga C.L. (2016). Triple negative breast cancers with amplification of JAK2 at the 9p24 loci demonstrate JAK2-specific dependence. Science translational medicine, 8(334), 334ra53.
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3

Generating Tumor Spheroids for Microenvironment Studies

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Six-well plates were incubated overnight on a rocker with 1.2% poly(2-hydroxyethyl methacrylate) (Cat# P3932; Sigma) diluted in ethanol. mCherry-expressing MCF-7 cells were plated in the coated wells, in DMEM/F12 medium supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin (all from Biological Industries), 0.4% BSA (Cat# 0332-TAM; Amresco, Solon, OH, USA), B-27 serum-free supplement (Cat# 17504044; Gibco, Life technologies, Grand island, NY, USA), 20 ng/ml rh-basic FGF (Cat# 100-18B; PeproTech), 20 ng/ml rh-EGF (Cat# 236-EG; R&D systems, Minneapolis, MN, USA), and 5 µg/ml insulin (Cat# I9278; Sigma). After 72 h, tumor spheroids were collected, centrifuged (1,200 rpm for 7 min, + 4°C) and resuspended in the different MSC-derived CM or the respective control media (as described above). Tumor spheroids were photographed daily using fluorescent microscopy.
Lerrer S., Liubomirski Y., Bott A., Abnaof K., Oren N., Yousaf A., Körner C., Meshel T., Wiemann S, & Ben-Baruch A. (2017). Co-Inflammatory Roles of TGFβ1 in the Presence of TNFα Drive a Pro-inflammatory Fate in Mesenchymal Stem Cells. Frontiers in Immunology, 8, 479.
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4

Immunophenotyping of IFNγ-Treated Cells

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IFNγ, InterMune (Brisbane, CA) and rhEGF R&D Systems, (Minneapolis, MN) were purchased. LMP-2 (clone SY-1), TAP1 (NOB1), TAP2 (NOB2), Tapasin (clone TO-3), calreticulin (TO-11), HLA-A (clone LGIII-147.4.1) and HLA-B/C (clone B1.23.2) were characterized previously (22 (link)). FITC-conjugated HLA-A/B/C (clone G46-2.6 or clone W6-32), APC-conjugated β-2m (clone 2M2), PE-conjugated IFNγ receptor α chain (clone GIR-208), p-STAT1 staining used PE-conjugated anti-p-STAT1 (Tyr701), APC-conjugated anti-STAT1 Ab (BD Biosciences), anti-STAT1 (C-24) polyclonal (pAb) (Santa Cruz Biotech, Santa Cruz, CA), anti-β-actin mAb (Sigma-Aldrich Inc, St. Louis, MO).
Srivastava R.M., Trivedi S., Concha-Benavente F., Hyun-bae J., Wang L., Seethala R.R., Branstetter BF I.V., Ferrone S, & Ferris R.L. (2015). STAT1-Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients. Cancer immunology research, 3(8), 936-945.
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5

Culturing Embryonic Mouse Neurofibroma Spheres

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Embryonic mouse spheres, dissociated from E12.5 DRG with 0.25% Trypsin 20 min. at 37°C (Mediatech; Herndon, VA) produced single-cell suspensions with narrow-bore pipettes and a 70 μm strainer (BD-Falcon). For mouse or human neurofibroma spheres, we chopped tissue into 1–3 mm3 pieces, plated in 20mL L-15 (Mediatech) plus 0.5 mg/mL collagenase type 1 (Worthington; Lakewood, NJ), and 2.5 mg/mL dispase protease type II (Cambrex; East Rutherford, NJ) at 37°C for 4–6 hours. We plated trypan blue negative cells (Stem Cell Technologies, Vancouver, BC) at 1 × 104 cells in 1 mL per well in 24-well low-binding plates in medium containing DMEM:F-12 (3:1) + 20 ng/ml rhEGF (R&D Systems), 20 ng/ml rh bFGF (R&D Systems), 1% B-27 (Invitrogen), 2 μg/ml heparin (Sigma). We maintained cultures at 37°C and 5% CO2 and counted floating spheres after 4–7 days. To passage, we centrifuged sphere cultures, dissociated and plated at 1 × 104 cells/ml in fresh sphere medium as described (Williams et al., 2008 (link)). For each experiment, we show a representative of 3 independent experiments.
Wu J., Keng V., Patmore D.M., Kendall J.K., Patel A.V., Jousma E., Jessen W.J., Choi K., Tschida B.R., Silverstein K.A., Fan D., Schwartz E.B., Fuchs J.R., Zou Y., Kim M.O., Dombi E., Levy D.E., Huang G., Cancelas J.A., Stemmer-Rachamimov A.O., Spinner R.J., Largaespada D.A, & Ratner N. (2016). Insertional mutagenesis identifies a STAT3/Arid1b/β-catenin pathway driving neurofibroma initiation. Cell reports, 14(8), 1979-1990.
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6

Culturing and Quantifying Salispheres

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UM-HMC-1,−3A,−3B (RRID:CVCL_Y473, RRID:CVCL_Y471, RRID:CVCL_Y472, respectively) cells (23 (link)) were cultured in ultra-low attachment plates or flasks (Corning; Corning, NY, USA) in DMEM/F12 (Invitrogen; Waltham, MA) supplemented with 1% N2 Supplement (Invitrogen), 1% GlutaMAX (Invitrogen), 1% antibiotic-antimycotic (Millipore Sigma, St. Louis, MO), 20 ng/mL rhEGF (R&D Systems; Minneapolis, MN), 20 ng/mL recombinant human basic FGF (R&D Systems), 10 ng/mL recombinant human insulin (Sigma-Aldrich, St. Louis, MO), and 1 μM Dexamethasone (Sigma-Aldrich). 4,000 cells/well were plated in 6-well ultra-low attachment plates and treated the following day with either MI-773 or APG-115, unless otherwise stated. Secondary salispheres were generated by dissociating primary spheres into single cell suspensions with Accutase (StemCell Technologies; Vancouver, Canada) and re-plating 4,000 cells/well in 6-well ultra-low attachment plates. Spheres were quantified 7–9 days after being plated, unless otherwise stated. Salispheres were defined as non-adherent spheres containing ≥30 cells, as observed through 100–200x magnification. Results are representative of a minimum of two independent experiments performed in triplicate experimental conditions.
Rodriguez-Ramirez C., Zhang Z., Warner K.A., Herzog A.E., Mantesso A., Zhang Z., Yoon E., Wang S., Wicha M.S, & Nör J.E. (2022). p53 inhibits Bmi-1-driven self-renewal and defines salivary gland cancer stemness. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(21), 4757-4770.
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7

Intestinal Organoid Culture Conditions

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After 21 days of differentiation in CK + AB or CK + DCI organoids were passaged and fed with SFFF or SFFF without FGF10 consisting of: Advanced DMEM/F12, 2 mM Glutamax, 1× B27 supplement, 100 U/mL penicillin-streptomycin, 15 mM HEPES, 0.05% BSA, 10 µM TGFβ inhibitor SB43152 (APExBIO, Cat#A8249), 1 µM p38 MAP kinase inhibitor BIRB796 (APExBIO, Cat#A5639), 3 µM CHIR99021, 50 ng/mL rhEGF (R&D Systems, Cat#236-EG) with (SFFF) or without the addition of 10 ng/mL FGF10 (produced in-house). Organoids were passaged every 7–14 days at a ratio of 1:6 by needle sheering93 .
Frum T., Hsu P.P., Hein R.F., Conchola A.S., Zhang C.J., Utter O.R., Anand A., Zhang Y., Clark S.G., Glass I., Sexton J.Z, & Spence J.R. (2023). Opposing roles for TGFβ- and BMP-signaling during nascent alveolar differentiation in the developing human lung. NPJ Regenerative Medicine, 8, 48.
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8

Derivation and Maintenance of Neural Stem Cells from Mouse ESCs

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NSCs were induced from
mouse ESCs using a previously reported method,
with some modifications.24 (link),25 (link) Briefly, embryoid bodies
(EBs) were formed with the hanging drop method using 7500 ESCs in
20 μL of medium in the absence of LIF for 3 days. The obtained
EBs were transferred to a low-adhesion plate (Corning Inc., Corning,
NY) and cultured in Neuron Culture Medium (Fujifilm Wako Pure Chemical
Corporation) supplemented with 20 ng/mL rhEGF (R&D Systems, Minneapolis,
MN) and 20 ng/mL rhFGF-2 (R&D Systems) for 96 h. Thereafter, the
EBs were transferred to matrigel (BD Biosciences, Franklin Lakes,
NJ)-coated dishes and incubated in NSC maintenance medium, MACS NeuroMedium
(Miltenyi Biotec, Bergisch Gladbach, Germany) containing 2% MACS NeuroBrew-21
(Miltenyi Biotec), 1% P/S, 20 ng/mL rhEGF, and 20 ng/mL rhFGF-2, for
20 days. Finally, NSCs that migrated radially outward from the EBs
were collected and cryopreserved until use in the in vitro NSC differentiation assay.
Otsuka S., Kawamura M., Fujino S., Nakamura F., Arai D., Fusetani N, & Nakao Y. (2022). Coronarin D, a Metabolite from the Wild Turmeric, Curcuma aromatica, Promotes the Differentiation of Neural Stem Cells into Astrocytes. Journal of Agricultural and Food Chemistry, 70(10), 3300-3309.
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9

Establishment of Mouse ZR^FUS Tumor Cell Line

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The mouse ZRFUS cell line was generated as described in our previous study (21 (link)). Briefly, mouse ZRFUS EPN tumors were collected based on the GFP signal, rinsed with PBS, and dissociated into single cells using the Brain Tumor Dissociation Kit (MACS, 130-095-942) following the manufacturer’s instructions. The cells were then grown in neural basal medium supplemented with sodium pyruvate, glutamine, B-27 (ThermoFisher, 12587010), N-2 (ThermoFisher, 17502048), rhEGF (R&D system, 236-EG), and rhFGF (R&D system, 4114-TC). Cells were cultured on Matrigel (Corning, 354277)-coated cultured dishes.
Sardar D., Chen H.C., Reyes A., Varadharajan S., Jain A., Mohila C., Curry R., Lozzi B., Rajendran K., Cervantes A., Yu K., Jalali A., Rao G., Mack S.C, & Deneen B. (2022). Sox9 directs divergent epigenomic states in brain tumor subtypes. Proceedings of the National Academy of Sciences of the United States of America, 119(29), e2202015119.
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10

TCF Activity Modulation by DKK3 Transfection

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In order to confirm TCF activity change by DKK3 transfection, a TOP/FOP Flash assay was performed as previously described13 (link). HSC-3 cells were seeded into 24-well plates at 1.0 × 105 cells/well and cultured for 24 h. Cells were then cotransfected with 0.2 μg of DNA constructs, 0.2 μg of reporter plasmid, and 0.2 μg of internal control pRG-TK, then cultured for a further 24 h. Luciferase activity was measured and normalized for transfection efficiency using a Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). Graphs show the average of three independent experiments with normalized transfection efficiency using Renilla luciferase. As a positive control, cells were stimulated by 100 ng/ml of rhEGF (R&D) for 24 h14 (link). To confirm the TCF reporter assay, one more experiment was done using Cignal™ Reporter Assay Kits (QIAGEN). As a control, cells were stimulated by 50 mM LiCl for 24 h.
Katase N., Nishimatsu S.I., Yamauchi A., Yamamura M., Terada K., Itadani M., Okada N., Hassan N.M., Nagatsuka H., Ikeda T., Nohno T, & Fujita S. (2018). DKK3 Overexpression Increases the Malignant Properties of Head and Neck Squamous Cell Carcinoma Cells. Oncology Research, 26(1), 45-58.
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