CD4+ CD25−CD62LhiCD44lo naive T cells were enriched from spleen using the naive CD4 T cells isolation kit (Miltenyi Biotec) with more than 92% purity. Naive T cells were then plated and cultured in 24-well plates. Naive T cells (106/0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D). In addition, recombinant cytokines and neutralizing antibodies were added to skew toward different Th cells in vitro. For Th1: mouse IL-12 (10 ng/mL), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience); for Th2: mouse IL-4 (10 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-12/23 p40 neutralizing antibody (10 μg/mL); for Th17 cells: mouse IL-6 (10 ng/mL; R&D System), human TGF-β1 (2 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience), and anti-IL-12/23 p40 neutralizing antibody (10 μg/mL; eBioscience) were added; for IL-27-inducing TR1, mouse IL-27 (20 ng/mL, R&D) were added; to skew toward in vitro-iTreg cells (iTreg), human TGFβ1 (2 ng/mL; R&D System) and mouse IL-2 (10 ng/mL; R&D System) were added at the same time.
Recombinant dll4
Manufactured by R&D Systems
Sourced in United States
Recombinant DLL4 is a soluble protein that corresponds to the extracellular domain of the human Delta-like 4 (DLL4) protein. DLL4 is a member of the Delta/Serrate/Lag-2 (DSL) family of Notch ligands.
Automatically generated - may contain errors
Lab products found in correlation
Human tgf β1, by R&D Systems (2 mentions)
Mouse il 2, by R&D Systems (2 mentions)
Naive cd4 t cells isolation kit, by Miltenyi Biotec (2 mentions)
Anti il 4 neutralizing antibody, by Thermo Fisher Scientific (2 mentions)
Anti ifnγ neutralizing antibody, by Thermo Fisher Scientific (2 mentions)
Anti il 12 23 p40 neutralizing antibody, by R&D Systems (2 mentions)
Anti il 12 23 p40 neutralizing antibody, by Thermo Fisher Scientific (2 mentions)
Mouse il 27, by R&D Systems (2 mentions)
Soluble anti cd28, by Thermo Fisher Scientific (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Mouse il 6, by R&D Systems (2 mentions)
Rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Silencer select, by Thermo Fisher Scientific (2 mentions)
Jag1 fc, by R&D Systems (2 mentions)
Huvecs, by Lonza (2 mentions)
Egm 2 singlequot kit, by Lonza (1 mentions)
Ebm 2, by Lonza (1 mentions)
4 aminophenylmercuric acetate apma, by Merck Group (1 mentions)
U2os cell, by American Type Culture Collection (1 mentions)
Batimastat bb94, by Merck Group (1 mentions)
8 protocols using recombinant dll4
1
Differentiation of Naive T Cells into Th Subsets
2
Culturing Primary Pulmonary Arterial Endothelial Cells
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Primary PAECs (Lonza, Frederick, MD, USA) were cultured for a maximum of 6 passages on cell culture flasks coated with type I collagen (50 μg/mL in 0.02 N acetic acid; Corning, Corning, NY, USA) in endothelial basal medium-2 (EBM™-2, Lonza, Frederick, MD, USA) supplemented with growth factors and 2% serum (EGM™-2 SingleQuot kit, Lonza, Frederick, MD, USA). Failed donor control (CTRL) and IPAH endothelial cells (ECs) obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) (see Supplementary Table S3 donor information) were plated on 0.1% gelatin (Sigma, St. Louis, MO, USA). In experiments studying exogenous DLL4 effects, cells were seeded on plates pre-coated with recombinant DLL4 (0.5 μg/mL in PBS overnight, R&D Systems; Minneapolis, MN, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO2 and 95% air.
3
Bone Marrow Isolation and Macrophage Differentiation
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Bone marrow was isolated from hind limbs of DLL4f/fLysMCreWT (Wt; n = 3) and DLL4f/fLysMCre+/0 (DLL4del; n = 3) mice. In short, the femur and tibiae were flushed with cold PBS. Bone marrow cells were cultured for 8 days in RPMI1640 cell culture medium (10% Fetal Calf Serum (FCS) (Bodinco BV, Alkmaar, The Netherlands), 1% penicillin/streptomycin, 1% L-Glutamine, 20mM HEPES) (GIBCO by Life technologies, Bleiswijk, the Netherlands) supplemented with 20% LCM (L929 cell conditioned medium which contains M-CSF) to differentiate into BMDM. All cells were cultured at 37°C in the presence of 5% CO2 atmosphere. Cells were seeded in a 24-wells plate (Greiner, 662102, Alphen a/d Rijn) (350,000 cells/well) and stimulated for 4 hours with LPS (100 ng/ml). For experiments with immobilized DLL4, cells were coated overnight with recombinant DLL4 (1 μg/ml; R&D Systems) and 0.2% gelatin in PBS at 4°C. Wells were rinsed once with PBS before plating Wt BMDM (350.000 cells/well) followed by 4 hrs incubation at 37°C. Tumor necrosis factor alpha (TNFα) protein was measured via ELISA (88-7324-88; Affymetrix, eBioscience, Vienna, Austria) according to the manufacturer’s protocol.
4
Notch Signaling in Synovial Fibroblasts
5
Differentiation of Naive T Cells into Th Subsets
6
Notch Signaling in Synovial Fibroblasts
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Synovial fibroblasts derived from RA synovial tissue were cultured in DMEM 10% fetal bovine serum as previously described 38 . For fibroblast Notch activation experiments, cell culture plates were coated with recombinant DLL4 or JAG1-FC (R&D systems) overnight at 4 degrees, at 5 μg/ml or at the concentration indicated for each experiment. Fibroblasts were then seeded on DLL4-, JAG1-FC- , or vehicle-coated plates for 72 to 120 hours. For cytokines and growth factors stimulation experiments, recombinant proteins were purchased (R&D systems) and reconstituted in DMSO or PBS per manufacturer’s instructions. Recombinant protein were diluted in media and then added to cultured fibroblasts at the following concentrations: TGFB1 10 ng/ml, BMP7 10 ng/ml, ACTIVIN A 10 ng/ml, WNT3A 100 ng/ml, WNT5A 100 ng/ml, EGF10 ng/ml, TNF 10 ng/ml, IFN-gamma or PDGF-BB 50 ng/ml. For flow cytometric analysis of Notch pathway in fibroblast-endothelial cell co-culture experiments, HUVECs (Lonza), passage 3 – 7, were cultured in a 1:1 ratio with synovial fibroblasts in EGM2 media for 72–96 hours in the presence or absence of 10 μM DAPT as indicated. For siRNA experiment, all siRNAs (Silencer Select) were purchased from Life Technologies. Fibroblasts were transfected with siRNA by reverse transfection at 30 nM using RNAi Max reagent (Life Technologies) as previously described 38 .
7
Cutaneous T-cell Lymphoma Cell Line Stimulation
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MyLa (mycosis fungoides cell line), HH (aggressive cutaneous T-cell lymphoma cell line) and Hut78 (Sézary syndrome cell line) cells were kindly provided by Dr Kazuyasu Fujii (Department of Dermatology, Kagoshima University, Kagoshima, Japan). The cells were cultured in RPMI 1640 containing 10% foetal bovine serum and supplements (penicillin G sodium, streptomycin sulphate and amphotericin B). CTCL cell lines were plated onto 96-well plates at 4×104 cells/200 μl per well. The cells were stimulated with recombinant DLL4 (1 or 5 μg/ml; R&D Systems), and incubated at 37°C and 5% CO2 for 24 or 48 h. Viable cells were counted by trypan blue exclusion.
8
DLL4, MMP-2, and MMP-9 Regulation in U2OS Cells
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Recombinant DLL4, MMP-2, and MMP-9 were purchased from R and D Systems. Batimastat (BB-94) was purchased from Sigma Aldrich. Compound E (GSI) was purchased from Fisher Scientific (Catalog # AAJ65131EXD). DECMA-1 antibody was purchased from Sigma-Aldrich (Cat# U3254, RRID:AB_477600 ). Z-DEV-FMK was purchased from R and D Systems (FMK004). E-cadherin primary antibody was purchased from Thermo Fisher Scientific (Cat# 33–4000, RRID:AB_2533118 ). β-tubulin primary antibody was purchased from Sigma-Aldrich (Cat#T8328, RRID:AB_1844090 ). U2OS cells were purchased from ATCC (Cat# HTB-96, RRID:CVCL_0042 ). MS5 and MS5-DLL4 cells were a kind gift from Dr. Stephen Blacklow. 4-aminophenylmercuric acetate (APMA) was purchased from Sigma-Aldrich. Herceptin was purchased from MedChemExpress (HY-P9907).
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