Severe asthmatic neutrophils suspended in the co-culture medium were pre-treated with 20 μg/mL of anti-human TGF-β1 antibody (MAB240, R&D Systems) for 2 h. Bronchial epithelial cells were then co-cultured with the neutrophils in the presence of anti-human TGF-β1 neutralizing antibody (20 μg/mL) for 48 h as previously described. Total RNA was isolated and purified to study changes in EMT marker expression.
Mab240
Manufactured by R&D Systems
Sourced in United Kingdom, United States
MAB240 is a monoclonal antibody product from R&D Systems. It detects and quantifies human Growth Differentiation Factor 15 (GDF-15) in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Tgf β1, by R&D Systems (1 mentions)
Mab002, by R&D Systems (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Sb431542, by Cayman Chemical (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Ab16828, by Abcam (1 mentions)
Ab9787, by Abcam (1 mentions)
Optiphot 2, by Nikon (1 mentions)
Immunofluorescence microscope, by Olympus (1 mentions)
Discovery ultra, by Roche (1 mentions)
Clone v 9, by Merck Group (1 mentions)
Alexa633 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Sc 52746, by Santa Cruz Biotechnology (1 mentions)
Axio imager m2 imaging system, by Zeiss (1 mentions)
Goat anti rabbit cy3, by Cell Signaling Technology (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Triton x 100, by Roche (1 mentions)
10 protocols using mab240
1
Neutralizing TGF-β1 in Asthmatic Neutrophil-Epithelial Co-culture
2
Exosome-mediated Downregulation of NK Cell NKG2D
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Assays in which normal human NK cells were co-incubated with exosomes to determine down-regulation of NKG2D on the surface of these cells were previously described (4 (link)). Briefly, NK cells (1 × 106) were co-incubated for 48 h with exosomes (50 μg) isolated from AML plasma. Then, flow cytometry for NKG2D expression was performed and data expressed as mean fluorescence intensity (MFI). NK cells incubated in medium were used as controls. Isolation of human NK cells from normal donor buffy coats using AutoMACS was also previously described (4 (link)). Expression patterns of TGF-β1 on the isolated exosomes, as seen in western blots, were correlated with the ability of these exosomes to inhibit NKG2D expression on human NK cells upon co-incubation. Antibodies to TGF-β (1 μg/mL, MAB240, R&D Systems) or isotype control antibodies added prior to co-incubation with exosomes were used to prevent down-regulation of NKG2D expression on NK cells. Recombinant human (rh) TGF-β1 (10 ng/mL) purchased from R&D Systems was used as a positive control.
3
Anti-TGF-β Treatment in Htra3 KO Mice
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For experiments with anti-TGF-β treatment, Htra3 KO mice were intraperitoneally injected daily with 1.5 mg/kg body weight of isotype IgG1 control antibody (#MAB002, R&D Systems) or anti-TGFβ1 antibody (#MAB240, R&D Systems) as previously described71 (link). Anti-TGF-β treatment was started 24 h before TAC operation.
4
Breast and Colon Cancer Cell Culture Protocol
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E0771 breast cancer cells (CH3 BioSystems; RRID:CVCL_GR23) and its derivative cell lines (E0771GFP, M11GFP, and M12GFP) were maintained in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% calf serum with iron (HyClone) and 1% penicillin-streptomycin. 67NR (from F. Miller, Karmanos Cancer Institute; RRID:CVCL_9723) and 4T1 [American Type Culture Collection (ATCC), catalog no. CRL-2539; RRID:CVCL_0125] breast cancer cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin at 37°C and 5% CO2. 293FT cells (ATCC, catalog no.PTA-5077; RRID:CVCL_6911), Ep5, and Ep5ExTu breast cancer cells (44 (link), 45 (link)), and MC38GFP colon cancer cells (46 (link), 47 (link)) were cultivated in Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. Cells were used at passage 4 to 15 after thawing and were mycoplasma negative, as tested with the MycoAlert Mycoplasma Detection Kit (Lonza).
For some experiments, cells were treated with doxycycline (1 μg/ml; Sigma-Aldrich, #9891), recombinant TGFβ1 (2 ng/ml; R&D Systems, #240-B), anti-TGFβ1 blocking antibody (6 μg/ml; R&D Systems, #MAB240; RRID:AB_358119), mouse immunoglobulin G1 (IgG1) isotype control (6 μg/ml; R&D Systems, #MAB002; RRID:AB_357344), or 10 μM TGFBR1 inhibitor SB431542 (Cayman Chemical, #13031), as indicated in the figure legends.
For some experiments, cells were treated with doxycycline (1 μg/ml; Sigma-Aldrich, #9891), recombinant TGFβ1 (2 ng/ml; R&D Systems, #240-B), anti-TGFβ1 blocking antibody (6 μg/ml; R&D Systems, #MAB240; RRID:AB_358119), mouse immunoglobulin G1 (IgG1) isotype control (6 μg/ml; R&D Systems, #MAB002; RRID:AB_357344), or 10 μM TGFBR1 inhibitor SB431542 (Cayman Chemical, #13031), as indicated in the figure legends.
5
Cytokine Expression in Root Canal Therapy
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All 12 rats from both the irradiated group and the non-irradiated group (n = 6 in each group) were euthanized at 2 or 3 weeks after root canal filling. Pentobarbital sodium was intraperitoneally administered, and perfusion fixation was performed with periodate lysine paraformaldehyde (PLP) fixative (Wako Pure Chemical Industries, Osaka, Japan). Mandibular samples containing the first molars were dissected, fixed in PLP for 12 h at 4°C, and decalcified in 10% EDTA containing 15% glycerol at 4°C. Serial sections of 5-μm thickness were stained with an enzyme antibody method using specific antibodies against interleukin-1β (IL-1β) (ab 9787; Abcam, Cambridge, England), TGF-β1 (MAB 240; R & D systems, Minneapolis, MN, USA), and FGF2 (ab 16828; Abcam) and then observed under a light microscope (Optiphot-2; Nikon, Tokyo, Japan).
6
Immunostaining of Neutrophil TGF-β1
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Some of the isolated neutrophils were used to prepare slides for immunostaining by cytospin. Neutrophils were suspended in RPMI medium at 500,000 cells per millilitre and 80 μL was used per slide. Neutrophils were then fixed with a 4% solution of paraformaldehyde for 20 min, and the slides stored at -20 °C. The Ventana DISCOVERY ULTRA automated slide preparation system was used to stain the neutrophils. The RUO DISCOVERY Universal protocol was applied where the primary antibody specific for human TGF-β1 (MAB-240, R&D Systems) at a 1:100 dilution was incubated for 60 min followed by fluorescently-tagged secondary antibodies (R&D Systems). Cell nuclei were stained with DAPI and the slides were observed with an Olympus immunofluorescence microscope.
7
Immunolabeling of Tissue Samples
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Prior to immunolabeling, tissue preservation, characterization and orientation were recorded by hematoxylin-eosin and Van Gieson stainings followed by histopathological analysis according to the EHRA classification [6] (link). Frozen sections were fixed for 10 min with 4 % paraformaldehyde. After washing in phosphate buffered saline (PBS) sections were incubated with 1 % bovine serum albumin for 30 min to block non-specific binding sites and then incubated with the primary antibodies. A monoclonal antibody against vimentin (clone V-9, Sigma) was directly conjugated with Cy3 (clone V9, Sigma) and a monoclonal antibody against TGF-β1 (MAB240, R&D) was used in dilutions as previously described [24] (link). Polyclonal primary antibodies against collagen V were purchased from Rockland. Anti-mouse or anti-rabbit IgG-conjugated with Cy3 or Cy2 (Biotrend) served as detection systems in single or double immunolabelings. The nuclei were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes). F-actin was fluorescently stained using TRITC-conjugated (Sigma) or Alexa633-conjugated phalloidin (Molecular Probes). Negative controls were obtained by omitting the primary antibody, in an otherwise similar protocol. Sections were embedded in Mowiol and coverslipped.
8
Immunohistochemical Analysis of Neuroinflammatory Markers
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For the detection of Tnfα and Tgfβ1 2 days after lesion with 6-OHDA, sections from SN and CPu were washed with PBS and blocked with PBS containing 10% normal goat serum and 0.1% TritonX-100 (Roche) for 1 h at RT. Afterwards, sections were incubated with primary antibodies anti-Tnfα (sc-52746, 1:100, Santa Cruz), anti-Tgfβ1 (MAB240, R&D System, 1:50, Wiesbaden-Nordenstedt) and anti-Iba1 (1:500, Wako Chemicals) at 4°C overnight, followed by an incubation with the corresponding Cy3-conjugated secondary antibodies (goat anti-mouse Cy3 1:100, goat anti-rabbit Cy3 1:200, Cell Signaling Technologies) for 1 h at RT. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, Roche). Fluorescence images were captured using the ZEISS AxioImager M2 imaging system (ZEISS, Göttingen, Germany).
9
Cytokine Profiling in Blood and Cells
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The concentration of cytokines in peripheral blood and cell culture supernatants was analyzed as previously described (43 (link), 44 (link)). Briefly, cytokines in the plasma of peripheral blood of children and cytokines released by non-stimulated and stimulated DCs after 24 h of stimulation with EcO83 or lipopolysaccharide (LPS) (1 µg/ml, cat. no. L2654-1MG; Sigma-Aldrich, St. Louis, MO, USA) were detected by ELISA. Reagents for IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, and transforming growth factor (TGF)-β detection were purchased from R&D Systems (Minneapolis, MN, USA) (IL-4: primary antibody MAB 604, biotinylated secondary antibody BAF 204, recombinant standard protein 204-IL; IL-5: MAB 405, BAM 6051, 205-IL; IL-6: MAB 206, BAF 206, 206-IL; IL10: MAB 2172, BAF 2017, 217-IL; IL-13: MAB 213, BAF 285, 285-IL; IFN-γ: MAB 2852, BAF 285, 285-IF; TGF-β: MAB 240, BAF 240, 240-B). Concentration of IL-10 in cell culture supernatants was determined by DUO SET DY217B (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
10
Immunofluorescence Staining of Osterix and TGF-β1
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The cells were fixed with 4% buffered formalin (cat. No. F-1268, Sigma-Aldrich) for 10 min and permeabilized with 0.1% Triton-X100 (cat. No. 22686, USB corporation, Cleveland, OH, USA) for 5 min. Nonspecific binding was blocked by incubating the cells with 2% horse serum (cat. No. SH30074, Hyclone, South Logan, UT, USA) for 30 min. The specimens were then incubated with primary antibody at 4 °C overnight. The primary antibodies used in the present study were rabbit anti-osterix (OSX) antibody (1000× dilution, cat. No. ab22552, Abcam, Cambridge, UK) and mouse anti-TGF-β1 antibody (10x dilution, cat. No. MAB240, R&D Systems). A biotinylated goat-anti rabbit antibody (1000× dilution, cat. No. sc-2040, Santa Cruz Biotechnology, Dallas, TX, USA) or a biotinylated goat-anti mouse antibody (1000× dilution, cat. No. B2763, LifetechnologiesTM, Eugene, OR, USA) were employed as secondary antibodies. Streptavidin-FITC (cat. No. S3762, Sigma-Aldrich) was used for fluorescence labeling. The specimens were incubated with secondary antibodies and Strep-FITC for 40 min each. The specimens were gently washed with PBS between each step. DAPI (cat. No. 5748, TOCRIS bioscience, Bristol, UK) was used to counterstain the nuclei. The specimens were then observed under an Apotome.2 (Carl Zeiss, Jena, Germany) fluorescence microscope.
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