RPE cells isolated from WT or immunoproteasome KO mice (L2 and L7M1) were immortalized as previously described [37 (link)]. RPE cells were cultured in Dulbecco’s Modified Eagle Medium as described previously [15 (link)], with 5% heat-inactivated fetal bovine serum (Atlanta Biologicals). For IGF-1 treatments, cells were serum starved for 24 hours, then treated with recombinant mouse IGF-1 (100 ng/mL, R&D Systems) and harvested at times indicated in the figures. Concentration of IGF-1 utilized in the study was determined by assessing time- and concentration-dependent phosphorylated-Akt (p-Akt) activation by Western blot in WT-RPE cells from 0–3 hours and 0–300 ng/mL IGF-1, respectively (Data not shown).
Recombinant mouse igf 1
Manufactured by R&D Systems
Sourced in United States
Recombinant mouse IGF-1 is a protein produced through recombinant DNA technology. It is a polypeptide growth factor that plays a role in cell growth and development.
Automatically generated - may contain errors
Lab products found in correlation
Erythropoietin, by Amgen (3 mentions)
Recombinant human insulin, by Merck Group (3 mentions)
Holo transferrin, by Merck Group (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Stemspan sfem, by STEMCELL (3 mentions)
Fetal bovine serum (fbs), by STEMCELL (3 mentions)
Igf 1, by R&D Systems (2 mentions)
Epoetin epo, by Amgen (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Cellmatrix type 1 a, by Nitta Gelatin (1 mentions)
Biacore t100 instrument, by GE Healthcare (1 mentions)
Series s sensor chip cm5, by GE Healthcare (1 mentions)
Recombinant mouse igfbp3, by R&D Systems (1 mentions)
Recombinant mouse scf, by R&D Systems (1 mentions)
Detoxified bsa, by STEMCELL (1 mentions)
Igf 1, by Abcam (1 mentions)
Cyclin d2, by Abcam (1 mentions)
Igf1r, by Abcam (1 mentions)
Cyclin d3, by Abcam (1 mentions)
12 protocols using recombinant mouse igf 1
1
Immortalized RPE Cells for IGF-1 Studies
2
Bioengineering Tooth Germs with IGF1
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Bioengineered tooth germs were generated as described previously2 (link). Molar tooth germs were dissected from the mandibles of ED14.5 C57BL/6 mice. To generate tissue recombinants, 5.0 × 104 epithelial and mesenchymal cells each in 0.05 µL were injected into 30-µL collagen gel drops of Cellmatrix type I-A (Nitta Gelatin, Osaka, Japan) using a Hamilton syringe (Osaka Chemical, Osaka, Japan). Recombinant mouse IGF1 (R&D Systems, McKinley, Minneapolis, MN, USA) (0.5, 1, 2, 4, and 8 µg/mL) was added to the gel drops in the IGF1-treated group.
3
Surface Plasmon Resonance of IGF1-IGFBP4 Interaction
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Surface plasmon resonance experiments were carried out on a Biacore T100 instrument (GE Healthcare). Recombinant mouse IGF1, 15 μg/ml in 10 mM sodium acetate pH 4.75, (R&D systems) was coupled to the EDC/NHS activated dextran matrix in flow cell 4 of a series S CM5 sensor chip (GE Healthcare), to a density of 130 response units. Flow cell 3 was left blank to serve as a reference cell. Subsequently, unreacted groups were blocked by a 7 min injection of 1 M ethanolamine, pH 8.0. To collect kinetic binding data, a twofold serial dilution of analyte (mouse wtIGFBP4/dBP4), ranging from 50 nM to 0.4 nM, in 10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl2 and 0.05% Tween-20, was injected over flow cells 3 and 4 at a flow rate of 30 μl/min. The association phase was 120 s, followed by a 240 s dissociation phase. Binding analysis was performed at 37 °C. The surfaces were regenerated by a 60 s injection of 10 mM glycine pH 2.0. Data were collected at a rate of 10 Hz. Recorded signals were subtracted the background signal, as determined by the response obtained from the reference cell. Global fitting of a 1:1 L model was performed, using the Biacore T200 Evaluation Software, version 1.0.
4
Enhancing Myogenic Differentiation with IGF-1
5
Mouse Metatarsal Culture Assay
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Mouse metatarsal culture was performed as previously described20 (link). Briefly, the middle three metatarsals were aseptically dissected from wild-type C57BL/6 mice. Bones were maintained in 500 μl α-MEM medium supplemented with 0.2% bovine serum albumin, 0.1 mM β-glycerophosphate, 50 μg ml−1 ascorbic acid, 1% Pen/Strep and 0.1% Fungizone. Bone were kept individually in 24-well plates in a humidified incubator at 37 °C, 5% CO2, for 3 days, medium was refreshed on day 2 of culture. DMSO, UNC1999 (2 μM), recombinant mouse Igf1 (100 ng ml−1, R&D systems, Minneapolis, MN), recombinant mouse Igfbp3 (50 nM, R&D systems) and/or recombinant mouse Igfbp5 (50 nM, R&D systems) were added at the beginning of culture and refreshed on day 2. Hypertrophic cell size in metatarsal bones was determined taking an area of 100 μm × 100 μm at the centre of the HZ, and measuring and averaging the height of every cell within the area.
6
Retroviral Transduction of Erythroid Progenitors
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MSCV-based retroviruses were produced and used to infect erythroid progenitors following a previously described protocol44 (link). Briefly, after isolation, lineage-negative fetal liver cells were plated in 24-well plates at 100,000 cells per well, covered by 1 ml virus containing supernatant, and centrifuged at 500 g for 90 min at 30 °C. After this spin-infection, the virus supernatant was replaced with erythroid maintenance medium (StemSpan-SFEM; StemCell Technologies) supplemented with 100 ng/ml recombinant mouse SCF (R&D Systems), 40 ng/ml recombinant mouse IGF1 (R&D Systems), 100 nM dexamethasone (Sigma), and 2 U/ml erythropoietin (Amgen) and cultured at 37 °C. GFP+ cells were sorted by flow cytometry after 16 h and cultured for another 48 h in erythroid differentiation medium (IMDM containing 15% (vol/vol) FBS (Stemcell), 1% detoxified bovine serum albumin (BSA; Stemcell), 500 μg/ml holo-transferrin (Sigma-Aldrich), 0.5 U/ml Epoetin (Epo; Amgen), 10 μg/ml recombinant human insulin (Sigma-Aldrich), and 2 mM L-glutamine (Invitrogen)) at 37 °C.
7
Erythroid Differentiation of Sf3b1 Mutant Cells
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Bone marrow cells that were isolated from Sf3b1K700E +/- mice and littermate controls were lineage depleted using the Lineage Cell Depletion Kit, mouse (Miltenyi Biotech) according to manufacturer’s protocols. Lineage negative cells were immediately transduced with lentiviral shRNAs targeting SF3A2 or controls (MOI −90) by spinfection at 2000 rpm for 90 min. The cells were cultured in erythroid maintenance medium (StemSpan-SFEM; StemCell Technologies) supplemented with 100 ng/mL recombinant mouse stem cell factor (SCF) (R&D Systems), 40 ng/mL recombinant mouse IGF1 (R&D Systems), 100 nM dexamethasone (Sigma), and 2 U/mL erythropoietin (Amgen) and cultured at 37°C for 36 hr. Following this, the cells were cultured for another 48 hr in erythroid differentiation medium (Iscove modified Dulbecco’s medium containing 15% (vol/vol) FBS (Stemcell), 1% detoxified BSA (Stemcell), 500 μg/mL holo-transferrin (Sigma-Aldrich), 0.5 U/mL Epoetin (Epo; Amgen), 10 μg/mL recombinant human insulin (Sigma-Aldrich), and 2 mM L-glutamine (Invitrogen)) at 37°C.
8
IGF1, Rapamycin, and Imidazole Ketone Erastin Administration in Mice
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For IGF1 administration, mice were subcutaneously treated with a dose of 200 µg/kg recombinant mouse IGF1 (R&D Systems, Minneapolis, MN, USA). For rapamycin (RAPA) and imidazole ketone erastin (IKE) administration, mice were intraperitoneally treated with a dose of 4 mg/kg RAPA (MedChem Express, Monmouth Junction, NJ, USA) or 40 mg/kg IKE (MedChem Express).
9
Comprehensive Analysis of IGF-1 Pathway Signaling
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Antibodies to GAPDH, IGF-1, IGF-1R, pho-IGF-1R, pho-IRS-1(Ser636/639), extracellular signal-regulated kinase (ERK), pho-ERK(Thr202/Tyr204), pho-AKT (Ser473), AKT, PARP, cyclin D1, cyclin D2, cyclin D3 and horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse and goat anti-rabbit) were purchased from Abcam (Cambridge, MA, USA), Millipore (Billerica, MA, USA), and Cell Signaling (Danvers, MA, USA). Recombinant mouse IGF-1, anti-human IGF-1 and anti-mouse IGF-1 enzyme-linked immunosorbent assay (ELISA) kit were obtained from R&D Systems (Minneapolis, MN, USA). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was from Promega (Madison, WI, USA).
10
Hypoxia-Reoxygenation H9c2 Cell Model
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For KLF3-AS1 overexpression, the pcDNA3.1 vector was used to construct the KLF3-AS1 plasmid by amplifying the coding sequence. KLF3-AS1 knockdown, miR-23c mimic, miR-23c inhibitor, and corresponding negative control (NC) were synthesized by GenePharma (Shanghai, China). Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the recommendations of the manufacturer.
H9c2 cells were maintained in DMEM deprived of glucose and serum in an anaerobic chamber with 1% O2, 94% N2, and 5% CO2 for 4 h. Then, the cells were maintained in DMEM supplemented with 10% FBS in a normal chamber with 95% O2 and 5% CO2 to establish the cellular hypoxia-reoxygenation (H/R) model in vitro. A Transwell (Corning, USA, 3412, 0.4 μm) coculture system was used to coculture H9c2 and MSC cells. H9c2 cells were seeded in 6-well plates at a density of 1 × 106 cells per well. Then, MSCs were seeded in polycarbonate Transwell inserts in 6-well plates. H9c2 and MSC cells were cocultured for 48 h at 37°C with 5% CO2. H9c2 cells were treated with an anti-IGF-1 neutralizing antibody (IGF-1 Ab, R&D Systems, USA) and 100 ng/mL recombinant mouse IGF-1 (R&D Systems, USA) to assess the role of IGF-1 in H9c2 cells.
H9c2 cells were maintained in DMEM deprived of glucose and serum in an anaerobic chamber with 1% O2, 94% N2, and 5% CO2 for 4 h. Then, the cells were maintained in DMEM supplemented with 10% FBS in a normal chamber with 95% O2 and 5% CO2 to establish the cellular hypoxia-reoxygenation (H/R) model in vitro. A Transwell (Corning, USA, 3412, 0.4 μm) coculture system was used to coculture H9c2 and MSC cells. H9c2 cells were seeded in 6-well plates at a density of 1 × 106 cells per well. Then, MSCs were seeded in polycarbonate Transwell inserts in 6-well plates. H9c2 and MSC cells were cocultured for 48 h at 37°C with 5% CO2. H9c2 cells were treated with an anti-IGF-1 neutralizing antibody (IGF-1 Ab, R&D Systems, USA) and 100 ng/mL recombinant mouse IGF-1 (R&D Systems, USA) to assess the role of IGF-1 in H9c2 cells.
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