Pdgf bb, by Fujifilm - Product details

1

Isolation and Culture of Aortic Smooth Muscle Cells

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Primary human aortic smooth muscle cells (Kurabo) were cultured in growth medium provided by the manufacturer and used for experiments at passage 5 (P5).
Mouse aortic smooth muscle cells were isolated from thoracic aortas of 8‐week‐old male mice as described previously 55. Briefly, the adventitia and endothelium were removed after digestion with collagenase type II (175 units/ml; Worthington) for 20 min. Subsequently, the media were digested with collagenase type II (175 units/ml) and elastase (0.5 mg/ml; Sigma) for 45–60 min. Isolated cells were cultured on collagen type I‐coated dishes (IWAKI) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂, and then, cells were used for experiments at passage 5 (P5).
For PDGF‐BB stimulation, cells were seeded in 6‐well plates at a concentration of 2.5 × 10⁵ cells per well and serum‐starved with differentiation medium provided by the manufacturer or DMEM containing 0.5% FBS for 24 h, and then subsequently stimulated with 10 ng/ml PDGF‐BB (WAKO) for 24 h.

Kimura M., Horie T., Baba O., Ide Y., Tsuji S., Ruiz Rodriguez R., Watanabe T., Yamasaki T., Otani C., Xu S., Miyasaka Y., Nakashima Y., Kimura T, & Ono K. (2020). Homeobox A4 suppresses vascular remodeling by repressing YAP/TEAD transcriptional activity. EMBO Reports, 21(4), e48389.

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2

Hypoxia and Fibrosis in PCLS

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Ex vivo culture of PCLS was performed at 37°C in 5% CO₂ for 96 h in the case of Poly(I:C) and/or IFNs and for 120 h in the case of fibrosis cocktail. IFN-γ (100 ng/ml, Fujifilm-Wako), IFN-α2 (100 ng/ml, R&D Systems), and Poly(I:C) (10 ng/ml, Tocris, Bristol, UK) were used to simulate RNA virus infections, such as SARS-CoV-2. The fibrosis cocktail consisted of 10 ng/ml platelet-derived growth factor (PDGF)-BB (Fujifilm-Wako), 10 ng/mL TNF-α (Fujifilm-Wako), 5 ng/mL transforming growth factor-β (TGF-β) (R&D systems), and 5 µM lysophosphatidic acid (LPA) (Focus Biomolecules, Plymouth Meeting, PA) and was replenished at 48 and 96 h (35 (link)). The O₂ concentrations for hypoxia and physioxia were used as 2 and 5%, respectively (39 (link), 40 (link)). PCLSs were incubated under hypoxia, physioxia, and normaxia (21% O₂) for 12, 24 and 48 h with or without Roxadustat (50 µM, Cayman, MI, USA) in hypoxia chamber (SV-140A, Blast, Tokyo).

Miura Y., Ohkubo H., Nakano A., Bourke J.E, & Kanazawa S. (2022). Pathophysiological conditions induced by SARS-CoV-2 infection reduce ACE2 expression in the lung. Frontiers in Immunology, 13, 1028613.

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3

Purification of Cell Adhesion Molecules

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Human plasma fibronectin was purified as described previously [36 (link)]. Tenascin-C was purified as described previously [37 (link)]. Peptide TNIIIA2 and FNIII14 has been described previously [11 (link),18 ]. PDGF-BB was purchased from Wako Pure Chemicals (Tokyo, Japan). PDGF-R kinase inhibitor AG1295 and Go6976, a specific inhibitor of PKC alpha isoforms, were obtained from calbiochem (San Diego, CA, USA). Antibody against PDGF-Rβ (2B3) was purchased from Cell Signaling Technology (Danvers, MA, USA). α-tubulin antibody (DM1A) and β1-integrin (N-20) antibody were purchased from Santa Cruz (Santa Cruz, CA, USA). Tenascin-C antibody (4F10TT) was purchased from IBL (Gunma, Japan). β1-integrin-neutralizing antibody (BV7) was purchased from abcam (Cambridge, UK).

Fujita M., Yamamoto T., Iyoda T., Fujisawa T., Nagai R., Kudo C., Sasada M., Kodama H, & Fukai F. (2019). Autocrine Production of PDGF Stimulated by the Tenascin-C-Derived Peptide TNIIIA2 Induces Hyper-Proliferation in Glioblastoma Cells. International Journal of Molecular Sciences, 20(13), 3183.

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4

Compound Sourcing for Cellular Assays

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Test compounds used in this study were
purchased from the following manufacturers: cyanidin 3-glucoside (Fuji
Film Wako Pure Chemical Corporation, Japan, #633-42451NS380101), cyanidin
chloride (Fuji Film Wako Pure Chemical Corporation, Japan, #030-21961),
platelet-derived growth factor-BB (PDGF-BB) (Fuji Film Wako Pure Chemical
Corporation, Japan, #166-19743), forskolin (Nacalai Tesque, Inc.,
Japan, #16384-84), isoproterenol (Nacalai Tesque, Inc., Japan, #19703-04),
and H89 (Cayman Chemical, United States, #10010556).

Krama A., Tokura N., Isoda H., Shigemori H, & Miyamae Y. (2022). Cyanidin 3-Glucoside Induces Hepatocyte Growth Factor in Normal Human Dermal Fibroblasts through the Activation of β2-Adrenergic Receptor. ACS Omega, 7(26), 22889-22895.

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5

Nesfatin-1 and PDGF-BB Sourcing

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Human nesfatin-1 was purchased from Sigma-Aldrich Japan (Product ID: SRP3291; Tokyo, Japan). Platelet-derived growth factor (PDGF)-BB was obtained from Wako (Osaka, Japan).

Mori Y., Shimizu H., Kushima H., Saito T., Hiromura M., Terasaki M., Koshibu M., Ohtaki H, & Hirano T. (2019). Nesfatin-1 suppresses peripheral arterial remodeling without elevating blood pressure in mice. Endocrine Connections, 8(5), 536-546.

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6

Efficient Cardiomyocyte Differentiation from iPSCs

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MCs were differentiated as described previously [2 (link)]. Briefly, human iPSCs were plated on dishes coated with the iMatrix-511 silk and cultured in StemFit AK02N for 2 days. The 10 μM ROCK inhibitor Y-27632 was used only on the first day. Then, the medium was replaced with mesoderm induction medium composed of D-MEM/F12 (Thermo Fisher Scientific, MA, USA) with 1% Glutamax (Thermo Fisher Scientific, MA, USA) and 1% B27 with 8 μM CHIR99021 and 25 ng/mL BMP4 (Fujifilm Wako Pure Chemical, Japan), followed by 3-day exposure of 2 ng/mL activin A and 10 ng/mL PDGFBB (R&D Systems, MN, USA). After 3 days, the mesoderm induction medium was replaced with MC induction medium consistent with StmePro-34 SFM (Thermo Fisher Scientific, MA, USA) supplemented with 10 ng/mL FGF2 (Fujifilm Wako Pure Chemical, Japan) and 10ng/mL PDGFBB for 3 days.

Tsuzuki S., Yamaguchi T., Okumura T., Kasai T., Ueno Y, & Taniguchi H. (2023). PDGF Receptors and Signaling Are Required for 3D-Structure Formation and Differentiation of Human iPSC-Derived Hepatic Spheroids. International Journal of Molecular Sciences, 24(8), 7075.

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7

Astrocyte Activation by Pericyte-Conditioned Media

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To prepare CM for treating astrocytes, pericytes were incubated in pericyte medium without FBS and pericyte growth supplement for 48 h but containing PBS or PDGF-BB (10 nM, Wako #16424031). Then, CM was collected [referred to as pericyte-CM (PCM)/PBS and PCM/PDGF-BB, respectively]. Primary astrocytes were treated with PCM and DMEM without serum at a ratio of 1:1 for 24 h. Control medium was prepared with pericyte medium without cultured pericytes and DMEM at a ratio of 1:1.

Shibahara T., Ago T., Nakamura K., Tachibana M., Yoshikawa Y., Komori M., Yamanaka K., Wakisaka Y, & Kitazono T. (2020). Pericyte-Mediated Tissue Repair through PDGFRβ Promotes Peri-Infarct Astrogliosis, Oligodendrogenesis, and Functional Recovery after Acute Ischemic Stroke. eNeuro, 7(2), ENEURO.0474-19.2020.

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8

Patient-Derived Glioma Cell Culture

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Patient-derived glioma cell cultures were generated as previously described⁴⁰. Briefly, tumor tissue was dissociated mechanically and enzymatically (Liberase DH, Roche) prior to separation of myelin and debris by sucrose centrifugation. Neurosphere-generating cultures were maintained in serum-free media supplemented with B27 (ThermoFisher), EGF, FGF, PDGF-AA, PDGF-BB (Shenandoah Biotechnology), and Heparin (StemCell Technologies). All cultures were validated and monitored by STR-fingerprinting (Supplementary Table 2) and verified to be mycoplasma-free within the previous 6 months (MycoAlert Plus, Lonza). SU-DIPG6 and SU-DIPG13 have been previously referred to as and are identical to SU-DIPG-VI and SU-DIPG-XIII, respectively. Clinical characteristics and STR fingerprints of all DIPG and pSCG cultures^{30 (link)} along with QCTB-R059^{4 (link)} used here have been previously reported. For all studies using human tissue, informed consent was obtained per guidelines of the approved Stanford Institutional Review Board protocol.

Mount C.W., Majzner R.G., Sundaresh S., Arnold E.P., Kadapakkam M., Haile S., Labanieh L., Hulleman E., Woo P.J., Rietberg S.P., Vogel H., Monje M, & Mackall C.L. (2018). Potent antitumor efficacy of anti-GD2 CAR T-cells in H3K27M+ diffuse midline gliomas. Nature medicine, 24(5), 572-579.

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9

Patient-Derived Glioma Cell Culture

Cited 2 times

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Patient-derived glioma cell cultures were generated as previously described⁴⁰. Briefly, tumor tissue was dissociated mechanically and enzymatically (Liberase DH, Roche) prior to separation of myelin and debris by sucrose centrifugation. Neurosphere-generating cultures were maintained in serum-free media supplemented with B27 (ThermoFisher), EGF, FGF, PDGF-AA, PDGF-BB (Shenandoah Biotechnology), and Heparin (StemCell Technologies). All cultures were validated and monitored by STR-fingerprinting (Supplementary Table 2) and verified to be mycoplasma-free within the previous 6 months (MycoAlert Plus, Lonza). SU-DIPG6 and SU-DIPG13 have been previously referred to as and are identical to SU-DIPG-VI and SU-DIPG-XIII, respectively. Clinical characteristics and STR fingerprints of all DIPG and pSCG cultures^{30 (link)} along with QCTB-R059^{4 (link)} used here have been previously reported. For all studies using human tissue, informed consent was obtained per guidelines of the approved Stanford Institutional Review Board protocol.

Mount C.W., Majzner R.G., Sundaresh S., Arnold E.P., Kadapakkam M., Haile S., Labanieh L., Hulleman E., Woo P.J., Rietberg S.P., Vogel H., Monje M, & Mackall C.L. (2018). Potent antitumor efficacy of anti-GD2 CAR T-cells in H3K27M+ diffuse midline gliomas. Nature medicine, 24(5), 572-579.

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10

Establishment and Characterization of Patient-Derived Glioma Neurosphere Models

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Patient-derived H3 K27M and H3WT glioma neurosphere lines were established at Dana-Farber Cancer Institute (BT869/BT869Luci; available from the Dana-Farber Cancer Institute Center for Patient Derived Models) and Hospital Sant Joan de Deu Barcelona (HSJD-DIPG007, HSJD-GBM001) as previously described (36 (link)–38 (link)). Neurosphere lines SU-DIPGXIIILuci, SU-DIPGXIIP*Luci, SU-DIPGXXV, SU-pcGBM2, and SU-DIPG48 were a gift from Michelle Monje at Stanford University, Stanford, California, USA. H3 K27M glioma cells were grown as neurospheres in tumor stem media base (38 (link)) supplemented with B27 minus vitamin A (Thermo Fisher Scientific), human growth factors (EGF, FGF, PDGF-AA, PDGF-BB from Shenandoah Biotechnology), and heparin (Stemcell Technologies) in ultra-low-attachment flasks. Indicated cell models expressing luciferase were generated as previously described (39 (link)). Neurosphere cultures were dissociated for passaging using Accutase cell detachment solution (Stemcell Technologies) for 3–5 minutes at 37°C. All neurosphere models were authenticated by high-resolution STR profiling (Molecular Diagnostics Core, Dana-Farber Cancer Institute). Whole-exome or whole-genome sequencing was conducted on neurosphere models to obtain copy number alterations.

So J., Mabe N.W., Englinger B., Chow K.H., Moyer S.M., Yerrum S., Trissal M.C., Marques J.G., Kwon J.J., Shim B., Pal S., Panditharatna E., Quinn T., Schaefer D.A., Jeong D., Mayhew D.L., Hwang J., Beroukhim R., Ligon K.L., Stegmaier K., Filbin M.G, & Hahn W.C. (2022). VRK1 as a synthetic lethal target in VRK2 promoter–methylated cancers of the nervous system. JCI Insight, 7(19), e158755.

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Pdgf bb

Lab products found in correlation

11 protocols using pdgf bb

Isolation and Culture of Aortic Smooth Muscle Cells

Hypoxia and Fibrosis in PCLS

Purification of Cell Adhesion Molecules

Compound Sourcing for Cellular Assays

Nesfatin-1 and PDGF-BB Sourcing

Efficient Cardiomyocyte Differentiation from iPSCs

Astrocyte Activation by Pericyte-Conditioned Media

Patient-Derived Glioma Cell Culture

Patient-Derived Glioma Cell Culture

Establishment and Characterization of Patient-Derived Glioma Neurosphere Models

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