Human ARPE-19 cell line and primary RPE cells were from ATCC (Manassas, VA) and Lonza (Walkersville, MD), respectively. Dulbecco’s Modified Eagle’s medium (DMEM), Ham’s/F12 medium, HEPES buffer, phosphate-buffered saline (PBS), penicillin-streptomycin, fetal bovine serum (FBS), 0.05% trypsin/EDTA, recombinant human CTGF, and Alexa Fluor-conjugated secondary IgG were from Invitrogen (Carlsbad, CA, USA). Recombinant human EGF, FGF-2, HGF, IGF-1, IL-6, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PTX3, and VEGF were from R & D Systems (Minneapolis, MN). Recombinant human G-CSF, IFN-γ, MCP-1, TGF-α, TGF-β1, TGF-β2, and TGF-β3 were from PeproTech (Rocky Hill, NJ). Hyaluronidase, protease inhibitors, bovine serum albumin (BSA), paraformaldehyde, methanol, Triton X-100, and Hoechst 33342 dye were from Sigma (St Louis, MO). α-SMA and BrdU antibodies were from Abcam (La Jolla, CA); β-catenin antibody was from BD Biosciences (San Jose, CA); Antibodies to lymphoid enhancer factor 1 (LEF1) and phospho-Smad2/3 were from Cell Signaling Technology (Danvers, MA). HMW HA, Healon® (~4,000 kDa, medical grade), was from Advanced Medical Optics (Santa Ana, CA). BCA Protein Assay Kit was from Pierce (Rockford, IL). HA quantitation kit was from Corgenix (Broomfield, CO). Plastic culture dishes were from Becton Dickinson (Lincoln Park, NJ).
Pdgf cc
Manufactured by R&D Systems
Sourced in Mongolia, United States
PDGF-CC is a recombinant human protein that belongs to the Platelet-Derived Growth Factor (PDGF) family. PDGF-CC is a homodimeric protein that can stimulate the proliferation and migration of various cell types, including fibroblasts, smooth muscle cells, and glial cells.
Automatically generated - may contain errors
Lab products found in correlation
Pdgf aa, by R&D Systems (2 mentions)
Pdgf ab, by R&D Systems (2 mentions)
Pdgf bb, by R&D Systems (2 mentions)
Phenol red free matrigel, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Tgf β3, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Phospho smad2 3, by Cell Signaling Technology (1 mentions)
Tgf β2, by Thermo Fisher Scientific (1 mentions)
Recombinant human gcsf, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Mcp 1, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Recombinant human egf, by R&D Systems (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
4 protocols using pdgf cc
1
In vitro RPE cell culture protocol
2
Mammary Stromal-Epithelial Organoid Co-Culture
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FACS-purified GFP+ cells from the lin- stromal fraction of PdgfrαCreR26mTmG mammary tissue were co-cultured with sorted tdTomato+ epithelial cells from Cre-R26mTmG mammary glands on top of a thin layer of phenol red-free matrigel (BD Biosciences) in a Lab-Tek II 4 chambered cover glass system (Nunc) in DMEM:F12 medium containing 5% FBS (GIBCO), insulin (Life Technologies), EGF (STEMCELL Technologies), bFGF (STEMCELL Technologies), hydrocortisone (STEMCELL Technologies), Rock inhibitor (Reagents Direct) in 5% oxygen conditions. After 4 days of organoid growth, cultures were treated with vehicle control (4 mM HCl + 0.1% BSA) or 100 ng/ml PDGFCC (R&D systems) and imaged overnight with Zen software using 20 × /0.8 Plan-Apochromat objective lens of an inverted motorized Zeiss AxioObserver microscope equipped with a stage top heated CO2 incubator.
3
Quantifying PDGFCC-induced Stromal Cell Migration
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In a modified Boyden Chamber assay, PdgfrαCreR26mTmG GFP+ stromal cells (40,000 cells/well) suspended in DMEM:F12 medium were placed on 6.5 mm transwell inserts with a 8.0 μm pore size and underside coated with 30μl of phenol red-free matrigel (BD Biociences) in 24 well plates (Corning). DMEM: F12 medium with vehicle control (4 mM HCl + 0.1% BSA) or 100 ng/ml PDGFCC (R&D systems) was added to the bottom chambers and cells incubated at 37 C for 4 h. Non-migrated cells were removed from the insert with a cotton swab, and migrated cells in the matrigel layer were fixed with paraformaldehyde and washed with PBS. GFP+ migrated cells were counted after imaging inserts on a cell imaging dish (Eppendorf) using a Zeiss LSM700 confocal microscope with Fluar 10X/0.50NA objective lens. Four microscope fields were counted per well and treatments performed in triplicate.
4
Chemotactic migration of cells
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We used recombinant human bone morphogenetic protein-2 (BMP-2; Medtronic, Minneapolis, MN, USA), hepatocyte growth factor (HGF; Peprotech, Rocky Hill, NJ, USA), insulin-like growth factor-1 (IGF-1; Peprotech), platelet-derived growth factor-AA (PDGF-AA; R&D Systems), PDGF-AB (R&D Systems), PDGF-BB (R&D Systems), PDGF-CC (R&D Systems), PDGF-DD (R&D Systems), stromal cell-derived factor-1α (SDF-1α; R&D Systems), transforming growth factor-β3 (TGF-β3; Miltenyi Biotec, Bergisch Gladbach, Germany). The medium containing 0.5% FBS in the lower chamber was supplemented with 100 ng/mL BMP-2, 100 ng/mL HGF, 100 ng/mL IGF-1, 10 ng/mL PDGF-BB, 100 ng/mL SDF-1α, or 10 ng/mL TGF-β3. After 48 h incubation, migrated cells were counted. The PDGF isoforms were compared by adding PDGF-AA, AB, BB, CC, or DD to the lower chamber at 100 ng/mL. Crenolanib (Selleck Chemicals, Houston, TX, USA), an inhibitor of PDGF receptor (PDGFR) α and β, was added to both the upper and lower chambers during the migration assay.
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