MDA-9/Syntenin (SDCBP) antibody was obtained from Abnova Inc. Antibodies for MST1, phosphor MST1, YAP, phosphor YAP, PDGFR-α, phospho-PDGFR-α and β-actin were purchased from Cell Signaling Technology. Fluorophore conjugated antibodies for various cell-surface markers, CD90, CD105, CD11b/mac, NK1.1 were obtained from eBioscience. CXCL5-AF700 were purchased from Novus Biologicals. Neutralizing antibodies for CXCL5 and PDGF-AA were obtained from R&D Systems. Human and mouse specific recombinant protein CXCL5, PDGF-AA, GM-CSF were procured from R&D Systems. MST overexpression and catalytically inactive pJ3M-Mst1 K59R were purchased from Addgene (43 (link)). YAP1 shRNA plasmids were obtained from GeneCopoeia, Inc. NF-κB inhibitor IKK2i was purchased from Sigma-Aldrich.
Pdgf aa
Manufactured by R&D Systems
Sourced in United States
PDGF-AA is a recombinant human Platelet-Derived Growth Factor AA homodimer. It is a member of the platelet-derived growth factor family, which is involved in the regulation of cell growth and division.
Automatically generated - may contain errors
Lab products found in correlation
Fibroblast growth factor 2 (fgf2), by R&D Systems (9 mentions)
Pdgf bb, by R&D Systems (7 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Pdgf ab, by R&D Systems (4 mentions)
N2 max, by R&D Systems (3 mentions)
Fibroblast growth factor (fgf), by R&D Systems (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Pdgf cc, by R&D Systems (2 mentions)
Image pro 5, by Media Cybernetics (2 mentions)
Mtt assay, by Merck Group (2 mentions)
Laminin, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (2 mentions)
40 protocols using pdgf aa
1
Antibody and Protein Sources for Cell Signaling
2
Quantifying hOPC Migration Responses
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hOPCs maintained in PDGF-AA and NT-3 were infected with either SULF2 KD or control scrambled lentivirus (1 multiplicity of infection). After 48 h, cells were seeded onto the upper surface of laminin-coasted transwell membranes (5 × 103/insert; 8-µm pore diameter, VWR). Recombinant human WNT3a (R&D Systems), PDGF-AA (Peprotech), or the combination of PDGF-AA and CXCL1 (R&D Systems) were added to the lower chamber (WNT3a, 5 ng/ml; PDGF-AA, 10 ng/ml; CXCL1, 5 ng/ml). At 16 h, cultures were fixed with 4% PFA and stained with DAPI (4′,6-diamidino-2-phenylindole). Cells on the upper surface of the transwell were removed and the remaining migrant cells were imaged (four ×10 fields/well; Olympus IX83) and counted in an unbiased manner using a Python OpenCV-based approach. Two-way ANOVA with Tukey’s HSD post hoc statistics were performed in R.
3
In vitro OPC Generation from EpiSCs
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EpiSC-derived OPCs were previously obtained using in vitro differentiation protocols and culture conditions and were a gift from Paul Tesar. Three independent derivations of OPCs generated from three separate EpiSC lines were used: OPC-5, OPC-1, and WT2.1. Data were generated using WT2.1 unless otherwise noted. OPCs were grown and expanded in flasks first coated with poly-ornithine (PO) (Sigma P3655; 100 μg ml−1) for 1 hour at 37 °C and dried then laminin-coated (Sigma L2020; 15 μg ml−1) for 1 hour at 37 °C. OPCs were grown in growth medium (DMEM/F12 (ThermoFisher, 11320-033) supplemented with N2-MAX (R&D Systems), B-27 (ThermoFisher), GlutaMax (Gibco), FGF (10 μg ml−1, R&D systems, 233-FB-025) and PDGF-AA (10 μg ml−1, R&D Systems, 233-AA-050) before harvesting and plating. Media was changed every 2 days. Cells were plated in differentiation-permissive media for experiments (DMEM/F12 supplemented with N2-MAX (R&D Systems), B-27 (ThermoFisher), GlutaMax (Gibco).
4
In vitro RPE cell culture protocol
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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).
5
OPC Differentiation Protocol with HDGF
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To induce OPC formation, dissociated primary neurosphere cells were plated at 39,500 cells/cm2 on pre-coated coverslips as described above (section C) in SFM supplemented with 2% B27, 10 ng/ml FGF and 10 ng/ml PDGF-AA (platelet derived growth factor AA, R&D) (herein referred to as OPC growth media [GM]) and cultured for 2-3 DIV. This culture system yields ∼94% Olig2+ and over 60% PDGFRα+ OPCs with no microglia contamination (Watson et al., 2021 (link)). To induce OPC differentiation, media was changed on 2-3DIV to SFM devoid of growth factors and supplemented with 2% B27 and 40 ng/ml T3 (3,3',5-Triiodo-L-thyronine, Sigma) (herein referred to as OPC differentiation media [DM]). Cells were cultured for additional 3 DIV in the presence of 10 ng/ml HDGF or VC (0.2 mM TRIS pH 7.5, 0.01 mM EDTA, 0.01 mM DTT, 0.1% glycerol in 1xPBS), or in the presence of 10 ng/ml HDGF and mouse IgG (sc-3877) or anti-C23 (Nucleolin) antibody (MS-3) (sc-8031) (Chen et al., 2015 (link); Lin et al., 2019 (link)).
6
MANF Expression in PDGF-AA Treated Microglia
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To determine whether the level of MANF is affected by various concentrations of PDGF-AA, the activated HAPI cells were exposed to recombinant rat PDGF-AA (R&D Systems, Minneapolis, MN, United States) at 0.1, 1, and 10 ng/mL for 48 h. Expression of MANF was detected by Western blot. To investigate the effect of PDGF-AA on LPS-stimulated microglia, the transfected HAPI cells were stimulated for 24 h using LPS (100 ng/mL). PDGF-AA at the selected concentration was added to the cell culture medium of HAPI cells for 48 h. The study design is briefly illustrated in Figure 1C .
7
PDGF-AA and Purmorphamine Stimulation Assay
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All cell lines were purchased from ATCC and cultured in DMEM containing glutamine and 10% FBS under standard conditions (humidity, 37 °C and 5% CO2) unless stated otherwise. Mouse IMCD3 cells were cultured in DMEM F-12 medium plus 10% FBS. For serum starvation, cells were washed 3 times in PBS and bathed in DMEM containing glutamine for overnight 16–20 hours (h) under standard conditions. For PDGF-AA (R&D Systems, 221-AA-010) stimulation, serum starved cells were treated with DMSO or 50 ng/ml of PDGF-AA for 0, 3 and 10 minutes (min). For serum stimulation, serum starved cells were treated with DMSO or 50 ng/ml of PDGF-AA for 0, 3 and 10 minutes (min). For purmorphamine (Sigma, SML0868) stimulation, serum starved cells were treated with DMSO or 5 µM purmorphamine for 0, 3 and 6 h.
8
Multivariate Characterization of Mesenchymal Stem Cells
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Several in vitro assays were performed to assess the cellular activities of MSCs. An MTT assay (Sigma-Aldrich) was used to assess cell proliferation, and a colony forming unit-fibroblast (CFU-F) assay was used to assess self-renewal. Multipotency (in vitro differentiation into chondrogenic, osteogenic, or adipogenic lineages) and transwell migration in response to platelet-derived growth factor (PDGF; 10 ng/mL PDGF-AA, R&D Systems, Minneapolis, MN, USA) were also assessed. Angiogenesis was quantified using Matrigel, and in vitro anti-inflammation was analyzed as described previously8 (link)–10 (link),20 (link),21 (link). The digital images generated in these assays were assessed quantitatively using Image-Pro 5.0 software (Media Cybernetics, Rockville, MD, USA).
9
Transdifferentiation of MEFs to OPCs
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MEFs were transdifferentiated to OPCs using the following protocol: cells were transfected (see below) and left in MEF culture medium for 3 days. After 3 days, the MEF culture medium was exchanged with MEF TD medium. TD medium contained the following DMEM/F12 (Invitrogen, no. 11320) supplemented with 1:100 N-2 (Life Technologies, no. 17502-048), 1:50 B-27 (Life Technologies, no. 17504-044), 2 mM GlutaMAX (Life Technologies, no. A1286001), 200 ng/mL SHH (R&D Systems, no. 461-SH-025/CF), 20 ng/mL FGF2 (R&D Systems), 20 ng/mL PDGF-AA (R&D Systems, no. 221-AA-010), and 2 μg/mL Laminin (Sigma).
To drive transdifferentiating MEFs to differentiate to MBP+ OL, 21 days after transfection TD cells were incubated in the following medium for 3 days: DMEM/F12 (Invitrogen, no. 11320) supplemented 1:100 N-2 (Life Technologies, no. 17502-048), 1:50 B-27 (Life Technologies, no. 17504-044), 2 mM GlutaMAX (Life Technologies, no. A1286001), 40 ng/mL T3 (Sigma), 200 ng/mL SHH (R&D Systems, no. 461-SH-025/CF), 100 ng/mL Noggin (R&D Systems, no. 3344-NG-050), 50 μM cAMP (Sigma, no. D0260-5MG), 100 ng/mL IGF (R&D Systems, no. 291-G1-200), 10 ng/mL NT3 (R&D Systems, no. 267-N3-005/CF). This is based on the protocol previously published by Najm et al. (2013) (link).
To drive transdifferentiating MEFs to differentiate to MBP+ OL, 21 days after transfection TD cells were incubated in the following medium for 3 days: DMEM/F12 (Invitrogen, no. 11320) supplemented 1:100 N-2 (Life Technologies, no. 17502-048), 1:50 B-27 (Life Technologies, no. 17504-044), 2 mM GlutaMAX (Life Technologies, no. A1286001), 40 ng/mL T3 (Sigma), 200 ng/mL SHH (R&D Systems, no. 461-SH-025/CF), 100 ng/mL Noggin (R&D Systems, no. 3344-NG-050), 50 μM cAMP (Sigma, no. D0260-5MG), 100 ng/mL IGF (R&D Systems, no. 291-G1-200), 10 ng/mL NT3 (R&D Systems, no. 267-N3-005/CF). This is based on the protocol previously published by Najm et al. (2013) (link).
10
Differentiation of Mouse OPCs from iPSCs
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Mouse OPCs were differentiated from mouse induced pluripotent stem cells (iPSCs) as previously described21 (link),49 (link). Briefly, iPSCs were removed from an irradiated mouse embryo fibroblast feeder layer with 1.5 mg/mL collagenase type IV (ThermoFisher, 17104019), dissociated with 0.25% trypsin-EDTA (ThermoFisher, 25200056), and seeded at 7.8×104 cells/cm2 on Costar Ultra-Low attachment plates (Sigma, CLS3471). Cells were cultured in media allowing for the expansion and maturation of OPCs for 9 days. On day 10, media was switched to OPC medium, comprised of N2B27 base medium, supplemented with 20 ng/mL FGF-2 (R&D Systems, 233-FB-010), and 20 ng/mL PDGF-AA (R&D Systems, 221-AA). N2B27 base medium consists of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; ThermoFisher 11320033), supplemented with 1X B-27 Supplement (ThemoFisher, 17504044), 1X N-2 MAX Supplement (ThermoFisher, 17502048), 1X GlutaMAX Supplement (ThermoFisher, 35050079). OPC medium was used over three passages to enrich for OPCs. OPC biological replicates were generated from independent mouse iPSC lines. Mouse iPSC-derived OPCs were used for all experiments unless otherwise noted.
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