To validate this assay, mouse embryonic fibroblasts (MEFs) were treated with 10ng/ml epidermal growth factor (EGF, Sigma-Aldrich, St. Louis, MO) for 15minutes. Then, MEFs were lysed and active Rac1 levels were measured by Rac1G-LISA Activation Assay Kit (enzyme-linked immunosorbent based assay) (Cytoskeleton Inc., Denver, CO) following manufacturer’s instruction. As a control, MEFs were incubated with 10ng/ml EGF and 100uM NSC23766, a specific Rac1 inhibitor (Tocris, Minneapolis, MN). Wild type, GOF and LOF Piezo1 BMDMs during phagocytosis (see above section on macrophage phagocytosis assay ) were lysed at different time points for detecting active Rac1 protein. Time zero (before phagocytosis) is the baseline signal and defined as zero. All data were normalized to time zero. Similarly, antibody specific for active Rac1 (1:200 dilution, NewEast Biosciences, King of Prussia, PA) was validated in MEFs by EGF and Rac1 inhibitor treatment. Wild type and GOF Piezo1 BMDMs were fixed by 4% PFA after 30min phagocytosis followed by antibody staining. Fluorescent images were captured by a Nikon Instruments A1R+ confocal microscope.
Epidermal growth factor (egf)
Manufactured by Bio-Techne
Sourced in Portugal, United Kingdom
EGF (Epidermal Growth Factor) is a protein that plays a crucial role in the regulation of cell growth, proliferation, and differentiation. It is a key component in cell signaling pathways and is widely used in various cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (3 mentions)
Te2000 u microscope, by Nikon (3 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
M csf, by Bio-Techne (2 mentions)
Pai 1, by Bio-Techne (2 mentions)
Phospho shp2, by Bio-Techne (2 mentions)
Interleukin 6 (il 6), by Bio-Techne (2 mentions)
Il 12p70, by Bio-Techne (2 mentions)
Tgf β1, by Bio-Techne (2 mentions)
Basic fibroblast growth factor (bfgf), by Bio-Techne (2 mentions)
Nsc23766, by Bio-Techne (1 mentions)
Rac1 g lisa activation assay kit, by Cytoskeleton (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Gastrin, by Merck Group (1 mentions)
Recombinant noggin protein, by Thermo Fisher Scientific (1 mentions)
Wnt3a conditioned medium, by R&D Systems (1 mentions)
Wnt c59, by Bio-Techne (1 mentions)
A83 01, by Bio-Techne (1 mentions)
10 protocols using epidermal growth factor (egf)
1
Rac1 Activity During Phagocytosis in Piezo1 BMDMs
2
Isolation and Culture of Pancreatic Cancer Organoids
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The isolation and culture of pancreatic cancer organoids was performed as previously described 13 (link). Human pancreatic cancer organoids were cultured in AdDMEM/F12 medium supplemented with GlutaMAX (Thermo Fisher Scientific), penicillin/streptomycin (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), N-acetyl-L-cysteine (1 mM, Sigma-Aldrich), Wnt3a-conditioned medium (50% v/v), RSPO1-conditioned medium (10% v/v, R&D Systems), recombinant noggin protein (100 ng/ml, PeproTech), recombinant epidermal growth factor protein (EGF, 50 ng/mL, PeproTech), gastrin (10 nM, Sigma-Aldrich), recombinant fibroblast growth factor 10 protein (FGF10, 100 ng/mL, PeproTech), nicotinamide (10 mM, Sigma-Aldrich), and A83-01 (0.5 μM, Tocris, Bristol, UK).
For ductal-like cancer cell differentiation, organoids were washed with basal medium and then cultured in AdDMEM/F12 with 50 ng/ml EGF, Wnt-C59 (100 nM, Tocris), DAPT (20 μM, Sigma-Aldrich), and B27 for 2 days.
For ductal-like cancer cell differentiation, organoids were washed with basal medium and then cultured in AdDMEM/F12 with 50 ng/ml EGF, Wnt-C59 (100 nM, Tocris), DAPT (20 μM, Sigma-Aldrich), and B27 for 2 days.
3
Growth Factor Infusion to Enhance Oligodendrogenesis
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Following a 5-week cuprizone challenge, the Nestin:YFP mice were given isoflurane anesthesia, and osmotic mini-pumps (ALZET® Model 2002) were implanted subcutaneously. These mini-pumps were used to infuse growth factors directly into the cerebrospinal fluid (CSF) through the cisterna magna at a flow rate of 0.25 μl/hour. The osmotic mini-pumps contained 100 μl of either EGF (66.7 μg/mL, Millipore Cat# 01-102), HB-EGF (20 μg/mL, R and D systems Cat# 259-HE-050/CF), EGF+HB-EGF (66.7 μg/mL and 20 μg/mL, respectively), or aCSF (vehicle control) (TOCRIS Bioscience Cat# 3528). This equates to 400 ng/day EGF, a dose previously demonstrated to induce oligodendrogenesis of neural stem cells in the V-SVZ 30 (link), and 120 ng/day HB-EGF, a dose previously demonstrated to restore neurogenesis in the V-SVZ in the aged brain 22 (link). The mini-pumps were removed after 2 weeks of implantation.
4
Cytokine and Growth Factor Assay Protocol
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Cytokine and Growth Factor Assay Protocol
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6
Investigating Growth Factor Signaling in SNB-19 Cells
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SNB-19 cells were serum starved for 24 h before being treated at indicated times with recombinant human proteins, including Gas6 (400 ng/ml), epidermal growth factor (EGF; 50 ng/ml), platelet-derived growth factor (PDGF; 10 ng/ml) or fibroblast growth factor (FGF; 10 ng/ml; Bio-techne, Abingdon, UK). In inhibition experiments, the EGFR small molecule inhibitor gefitinib (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or the Axl small molecule inhibitor BGB324 (BerGenBio AS, Bergen, Norway) were pre-incubated with cells for 1 h prior to EGF stimulation.
7
Spheroids Assay for Synergistic Drug Effects
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HCT116 cells were resuspended in serum-free stem cell culture media consisting of DMEM/F12 supplemented with 10 ng·mL−1 of bFGF, 20 ng·mL−1 of EGF from Biotechne (Citomed Lda, Lisboa, Portugal), 1 × B27 from Life Technologies (Porto, Portugal), and 5 μg·mL−1 of insulin (Sigma-Aldrich). HCT116 cells were plated in 24-well ultra-low attachment plates (one spheroid per well; Corning Inc., Sigma-Aldrich), at a density of 1 × 103 cells/well [48 (link)]. To assess the synergistic effect of SLMP53-1 with DCA in spheroids development, colonospheres were allowed to form for 3 days, followed by treatment with 20 μM SLMP53-1 and/or 12 mM DCA, for an additional 9 days. During this period of time, new medium with the drugs (or DMSO only) was added to the wells each three days. Spheroids were photographed by using an inverted Nikon TE 2000-U microscope at ×100 magnification, with a DXM1200F digital camera and NIS-Elements microscope imaging software (VWR). Determination of spheroids diameter was performed by using Image J software.
8
HCT116 Cell Spheroid Formation and Treatment
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HCT116 cells were resuspended in serum-free stem cell culture media consisting of Dulbecco’s modified Eagle medium supplemented with 10 ng/mL bFGF and 20 ng/mL EGF (Bio-techne, Citomed Lda, Lisboa, Portugal), 1× B27 (Life Technologies), and 5 μg/mL insulin (Sigma-Aldrich). Cells were plated in 24-well ultra-low attachment plates (Corning Inc.; one spheroid/well) at 1 × 103 cells/well. Treatments with Roy-Bz or vehicle were performed 3 days after spheroid formation for up to 96 h, or at the seeding time for 48 h. Spheroid formation was monitored using an inverted Nikon TE 2000-U microscope at ×100 magnification with a DXM1200F digital camera and using Nikon ACT-1 software. Spheroid diameters were quantified using the ImageJ software.
9
Seeding and Expansion of NIKS Cells
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NIKS and all NIKS‐derived cells were seeded at a cell density of 1 × 105 cells per well, in F medium (Sigma, Haverhill, UK) without EGF (236EG‐01 M; Bio‐Techne, Abingdon, UK), on 1 × 105 γ‐irradiated J2‐3 T3 in six‐well plates. One day later (day 1), the plating efficiency was estimated and cells were fed with fresh F medium supplemented with 10 ng/ml EGF (Bio‐Techne). To harvest the cells, feeder cells were first dislodged by a short trypsinization step and NIKS keratinocytes were then collected after a second trypsinization and counted using a Z1 Coulter particle counter (Beckman, Takeley, UK). Unless otherwise specified, total cell numbers/ml for each triplicate were assessed on days 1, 3, 4, 5, 7, and 9.
10
Mammosphere Assay with BBIT20, CDDP and Olaparib
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