Plugs of 0.5 mL Growth Factor Reduced matrigel (356231, BD Biosciences, San Jose, CA, USA), supplemented with either 600 ng/mL recombinant murine FGF-2 (450-33, Peprotech, London, UK) and 3 U/mL heparin (Biochrom AG, L6510) or 200 ng/mL recombinant murine VEGF165 (450-32, Peprotech, London UK) and 10 U/mL heparin, were s.c. injected into the flank of either WT or Cx37−/− mice. Matrigel plugs lacking growth factors were used as negative controls. WT mice were also injected with Growth Factor Reduced matrigel supplemented with 200 ng/mL VEGF, 10 U/mL heparin and 300 µM of either the 37,43Gap27 peptide or its scrambled version. The plugs were removed 1 week later for evaluation of vascularization, as published [28 (link)]. Specifically, the macroscopic observations of all implanted plugs showed the presence of blood vessels, and the color difference of the plugs implanted in control mice and Cx37−/− mice paralleled a different hemoglobin content, providing an indirect, still reliable and commonly used indicator of angiogenesis [28 (link)].
Recombinant murine fgf 2
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Recombinant murine FGF-2 is a laboratory reagent produced using recombinant DNA technology. It is a member of the fibroblast growth factor family and is derived from the mouse species. The core function of this product is to promote cellular growth and differentiation in various in vitro and in vivo applications.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Thermo Fisher Scientific (2 mentions)
Recombinant murine vegf165, by Thermo Fisher Scientific (2 mentions)
Heparin, by Harvard Bioscience (2 mentions)
Growth factor reduced matrigel, by BD (2 mentions)
Collagenase, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mi 503, by MedChemExpress (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Hbss antibiotic, by Thermo Fisher Scientific (1 mentions)
Ham f12 nutrient mixture, by Thermo Fisher Scientific (1 mentions)
Mek inhibitor, by Selleck Chemicals (1 mentions)
Pd0325901, by Selleck Chemicals (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Recombinant murine pdgf bb, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Merck Group (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
5 protocols using recombinant murine fgf 2
1
Matrigel Plug Angiogenesis Assay
2
Murine Uterine Stromal Cell Isolation and Decidualization
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Three to four pseudopregnant day 4 mouse uterine horns were cut into small pieces (2–3 mm). Tissue pieces were first digested in 3 ml fresh medium (HBSS antibiotic; Gibco) containing 6 mg/ml dispase (Gibco) and 25 mg/ml pancreatin (Sigma), and then incubated in fresh medium containing 0.5 mg/ml collagenase (Sigma) at 37 °C for 30 min. The digested cells were passed through a 70 μm filter to obtain the stromal cells. Cells were plated at 60 mm dishes or 6-wells plates, containing phenol red-free Dulbecco modified Eagle medium (DMEM) and Ham F12 nutrient mixture (1:1) (Gibco) with 10% charcoal-stripped fetal bovine serum (CS-FBS) and antibiotic. Two hours later, the medium was replaced with fresh medium (DMEM/F12, 1:1) with 10% CS-FBS. The next morning, the medium was replaced with DMEM/F12 containing 1% C-FBS, E2 (Sigma, 10 nM), P4 (Sigma, 1 μM) and antibiotic to induce decidualization. The media was changed every 48 h. For the treatment of primary uterine stromal cells, MI-503 (2 μM; MedChemExpress), recombinant murine FGF2 (PeproTech, 20 ng/ml), recombinant mouse PTX3 (R&D systems, 100 ng/ml) and MEK inhibitor PD0325901 (Selleck, 5 μM) were used.
3
Myh11-Lin(+) ASCs Corneal Endothelial Differentiation
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Myh11-Lin(+) ASCs were uplifted and cultured on FNC Coating Mix (NC9971265; ThermoFisher). Once Myh11-Lin(+) ASCs reached greater than 90% confluency, the standard media was replaced by corneal endothelial differentiation media as previously described.18 Briefly, Myh11-Lin(+) ASCs were exposed for 3 days to dual Smad induction media, which consisted of Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 20% KnockOut serum replacement (10828028; ThermoFisher), 1% nonessential amino acids (11140076; ThermoFisher), 500 ng/mL Noggin (SRP3227; Sigma, St. Louis, MO, USA) 10 μM SB431542 (S4317; Sigma), 1 mM L-glutamine (25030081; ThermoFisher), 0.1 mM betamercaptoethanol (21985023; ThermoFisher), and 8 ng/mL recombinant murine FGF-2 (450–33; PeproTech, Rocky Hill, NJ, USA). On day 3, the dual Smad induction media was replaced with dual Smad induction media supplemented with 0.1X B27 supplement (17504044; ThermoFisher), 10 ng/mL recombinant mouse Dkk-2 (2435-DKB/CF; R&D Systems, Minneapolis, MN, USA), and 10 ng/mL recombinant murine PDGF-BB (315–18; PeproTech). This media solution was changed every 3 days over the course of 14 days. After the course of 14 days, cells were fixed and stained using methods described above.
4
Angiogenic Factors Induce Vascularization
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Plugs of 0.5 mL Growth Factor Reduced matrigel (BD Biosciences, 356231) supplemented with either 500 ng/ml recombinant murine FGF-2 (Peprotech, 450–33) and 3 U/ml Heparin (Biochrom AG, L6510) or 200 ng/ml recombinant murine VEGF165 (Peprotech, 450–32) and 10 U/ml Heparin [53 (link)], were s.c. injected into the flank of either WT or Cx40−/− mice. Matrigel lacking growth factors were used as negative control. WT mice were also injected with Growth Factor Reduced matrigel supplemented with 200 ng/ml VEGF, 10 U/ml Heparin and 300 μM of either peptide 40Gap27 or its scrambled version. The plugs were removed 1 week after injection, for evaluation of vascularization.
5
Preparation of Growth Factor-Adsorbed BGMs
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BGMs were prepared according to the original report (Tabata et al., 1999 (link)). For single testis treatment, freeze-dried BGMs (1 mg) were reconstituted with 10 μL of 1 mg/mL recombinant murine FGF2 or GDNF (PeproTech, London, UK) in distilled-deionized-autoclaved water. In this manner, growth factor solutions were completely absorbed in BGMs, demonstrating that the growth factors were entirely contained within BGMs. After overnight adsorption of growth factors at 4°C, the resultant BGMs were suspended in 15 μL of autoclaved physiological saline for transplantation.
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