NSCLC cells (A549, EKVX, NCI-H23, NCI-H226, NCI-H322M, and NCI-H522) were obtained from the NCI-Fredrick Cancer Center and were routinely cultured in RPMI-1640 medium (Gibco BRL) for 4–6 passages. Short tandem repeats profiling was used to confirm the identity of the NSCLC cells (Supplementary Table S1, S2 ). Immunodeficient mice (nu/nu; Harlan Sprague–Dawley, Inc. Indianapolis, IN) were maintained under pathogen-free conditions at the animal resources center (ARC) at Asan Medical Center, and all animal studies were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. Mice used for xenograft experiments were euthanized by CO2 inhalation (30% vol/min). Human recombinant epidermal growth factor (EGF, 236-EG, R&D systems, USA) protein, C646 (SML 0002-5MG, SIGMA Life Science), SF1670 (SML 0684-5MG, SIGMA Life Science), and MRS 2500(2159, TOCRIS Bioscience) were used for experimental treatments
Egf 236 eg
Manufactured by R&D Systems
Sourced in United States
EGF (236-EG) is a recombinant human epidermal growth factor (EGF) protein. EGF is a growth factor that stimulates cell growth, proliferation, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Cell culture grade, by Merck Group (3 mentions)
Ebm 2 basal medium, by Lonza (3 mentions)
Tak 165, by Bio-Techne (3 mentions)
Egm 2 singlequot kit, by Lonza (3 mentions)
Dispase 2, by Roche (3 mentions)
Collagenase 2, by Worthington (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Ast 1306, by Selleck Chemicals (2 mentions)
D glucose and dnase 1, by Merck Group (2 mentions)
Gi254023x, by Bio-Techne (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
N acetyl l cysteine, by Fujifilm (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Unc1062, by Aobious (1 mentions)
Bs3 reagent, by Thermo Fisher Scientific (1 mentions)
Relafin, by R&D Systems (1 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
13 protocols using egf 236 eg
1
Non-small Cell Lung Cancer Cell Lines and Xenograft Model
2
3D Culture of PDAC Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five patient-derived PDAC organoids (KYK070, KYK002, KYK023, KYK090 and KYK093) were cultured three-dimensionally in growth factor reduced Matrigel (354230; Corning, Corning, NY, USA) with complete organoid media containing advanced DMEM/F12 (12634-010; Life Technologies) supplemented with 10% Afamin/Wnt3a CM (J2-001; Medical & Biological Laboratories), 10% R-spondin1-conditioned medium from Cultrex R-spondin1 Cell and Reagent (3710-001-01; R&D Systems, Minneapolis, MN, USA), 10 mM HEPES (15630-080; Life Technologies), 1% GlutaMax (35050-061; Life Technologies), 2% B27 (17504044; Life Technologies), 10 nM gastrin-I (G9020-1MG; Merck KGaA), 500 mM N-acetyl-L-cysteine (017-05131; FUJIFILM Wako Pure Chemical Corporation), 10 ng/ml EGF (236-EG; R&D systems), 100 ng/ml noggin (6057-NG; R&D Systems), 1 mM A83-01 (SML0788-5MG; Merck KGaA), 100 ng/ml FGF-10 (060-04401; FUJIFILM Wako Pure Chemical Corporation) and 10 mM nicotinamide (N0636; Merck KGaA). Media were replaced every two to three days. All organoids were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
3
Prostatosphere Formation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prostatosphere formation assays were performed using a slight modification of previously described techniques [39 (link), 40 (link)]. Cells were plated in DMEM F-12 (11320-033, Life Technologies) containing 10 ng/mL bFGF (233-FB/CF, R&D Systems, Minneapolis, MN), 20 ng/mL EGF (236-EG, R&D Systems), 5 mg/mL insulin (3435, R&D Systems), and 0.4% (v/v) bovine serum albumin (BSA) (5217, R&D Systems) supplemented with 1% (v/v) knockout serum replacement (10828-028, Life Technologies) at 500-3,000 cells per well in 6-well ultra low attachment plates. In some case, the cultures were treated with the Mer inhibitor UNC1062 (AOB4488, AOBIOUS, Gloucester, MA). Prostatosphere formation (cell clusters of 10 cells or greater) was observed at 7–10 days under a light microscopy.
4
EGFR Dimerization and Internalization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After starvation for 12 h, wild-type HCC cells were stimulated with 10 μg/ml recombinant Elafin (rElafin, R&D Systems, MN, USA) or 20 ng/ml EGF (236-EG, R&D Systems, MN, USA) or with BSA (Biofroxx, Germany) as the control for 30 min. After that, the cells were treated with the crosslinking reagent BS3 reagent (Thermo Fisher, MI, USA) at a concentration of 3 mM for 30 min at room temperature. Then, 40 μL 1 M Tris-HCl pH 7.5 was added for an additional 15 min to terminate the reaction. Finally, the cells were lysed and subjected to western blotting to assess EGFR dimerization. The dimers were represented by bands at approximately 300 KD, while EGFR monomers were represented by bands at 170 KD.
To assess the internalization of EGFR, after starvation for 12 h, the cells were stimulated with 10 μg/ml recombinant Elafin or 20 ng/ml EGF or BSA for 30 min. Then, the cells were subjected to immunofluorescence (IF) to assess the cell surface expression of EGFR.
To assess the internalization of EGFR, after starvation for 12 h, the cells were stimulated with 10 μg/ml recombinant Elafin or 20 ng/ml EGF or BSA for 30 min. Then, the cells were subjected to immunofluorescence (IF) to assess the cell surface expression of EGFR.
5
Stemness Marker Antibody Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti CD133 (ab222782; 1:1000) and anti Nestin (ab134017; 1:500) antibodies were purchased from Abcam (Cambridge, MA). Anti Sox2 clone D6D9 (3579; 1:1000) and anti Oct-4A clone C30A3 (2840; 1:500) antibodies were purchased from Cell Signaling (Danvers, MA). Anti L1CAM (AF277; 1:1000) and anti-NFASC (AF2325; 1:500) were purchased from R&D Systems (Minneapolis, MN). Anti NCAM2 (sc-136328; 1:500) was purchased from Santa Cruz Biotechnology (Dallas, TX). Anti β-actin clone AC-40 (GTX11003; 1:10000) was purchased from Genetex (Irvine, CA). Anti CD11b/c clone OX-42 (201807; 1:100) was purchased from BioLegend (San Diego, CA). Anti CD133 clone TMP4 (46-1338-42: 1:200) antibody, Live/Dead fixable red stain (1:200; L34972) and Live/Dead fixable yellow stain (1:200; L34968) were purchased from Thermo Fisher Scientific. Secondary anti-rabbit IgG HRP-linked (7074; 1:10000) and anti-mouse IgG HRP-linked (7076; 1:10000) antibodies were purchased from Cell Signaling. Recombinant bFGF (233-FB) and EGF (236-EG) were purchased from R&D Systems (Minneapolis, MN).
6
Antibody-based Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against PCNA (2586), phosphor-MEK1/2 (9154), phosphor-p90RSK (11989) and phosphor-MSK1 (9595) were obtained from Cell Signaling Technology (CST, MA, USA). Antibodies against AdipoR1 (ab126611), p90RSK (ab32114), cyclinD1 (ab134175), CDK4 (ab108357), CDK6 (ab124821), total-ERK1/2 (ab184699), phospho-ERK1/ (ab76299) and total-MEK1/2 (ab178876) were obtained from Abcam (Cambridge, UK). Recombinant human adiponectin (1065-AP) and recombinant human epidermal growth factor (EGF, 236-EG) were obtained from R&D System.
7
Endothelial Cell Culture Protocol
8
Isolation and Culture of Glioma Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glioma stem cells (GSC) were derived from human primary glioblastoma tissues, and the culture method is detailed in Gao et al17 (link) and Bhat et al.18 (link) Freshly resected glioma tissue was cut into small pieces and digested by trypsin. Then, the dissociated single cells and remaining tissues were cultured with DMEM/F12 media (10565018; Gibco) supplemented with B27 (17504044; Gibco), 20 ng/mL EGF (236-EG; R&D Systems, Minneapolis, MN), and 20 ng/mL bFGF (233-FB; R&D Systems). One to 2 weeks later, the floating tumor neurospheres were collected and subcultured in the media mentioned above. Regarding the sphere-formation assay, after digesting tumor neurospheres by Accutase (A6964; Sigma-Aldrich), 1000 single GSC cells per well were cultured with or without NC treatment. After 1 week, the spheres were observed with the Leica microscope.
9
Isolation and Culture of Endothelial Cells
10
Recombinant Human ErbB Ligands and Inhibitors
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!