Cells were collected via EDTA-PBS (0.4% EDTA) treatment without trypsin on ice. After washing with PBS, cells were blocked with PBS containing 2% bovine serum albumin, 5% normal goat serum, and 5 mM EDTA on ice for 30 min. Cells were incubated with the primary antibody anti-AREG (1:100; R&D Systems, Minneapolis, MN) on ice for 30 min, washed with PBS, and then treated with the secondary antibody on ice for 15 min. After two PBS washes, the level of AREG bound on cell membrane was measured with a FACSCalibur (Becton-Dickinson, San Jose, CA).
Anti areg
Manufactured by R&D Systems
Sourced in United Kingdom, United States
Anti-AREG is a recombinant protein that acts as an antibody against the Amphiregulin (AREG) molecule. AREG is a member of the epidermal growth factor (EGF) family and is involved in various cellular processes. The Anti-AREG product can be utilized in research applications to study the role and function of AREG.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (1 mentions)
Anti cd127, by BioLegend (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Thermo Fisher Scientific (1 mentions)
Anti nk1, by Thermo Fisher Scientific (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Ghost dye, by Cytek Biosciences (1 mentions)
Anti ly6c, by Thermo Fisher Scientific (1 mentions)
Anti il 17, by Thermo Fisher Scientific (1 mentions)
Anti tcrβ, by Thermo Fisher Scientific (1 mentions)
Anti cd103, by Thermo Fisher Scientific (1 mentions)
Anti cd62l, by Thermo Fisher Scientific (1 mentions)
Anti cd5, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Anti st2, by Thermo Fisher Scientific (1 mentions)
Anti cd45r b220, by Thermo Fisher Scientific (1 mentions)
Anti klrg 1, by Thermo Fisher Scientific (1 mentions)
Anti ctla 4, by Thermo Fisher Scientific (1 mentions)
Anti pd 1, by Thermo Fisher Scientific (1 mentions)
Anti foxp3, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti areg
1
Cell Surface AREG Quantification
2
Lung and Tumor Immune Cell Isolation
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Lymphocytes were isolated from lung and tumor tissue by digestion with collagenase A (1 mg/ml; Roche) and DNase I (0.5 µg/ml; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% l -glutamine, 1% penicillin-streptomycin, and 10 mM Hepes) for 30 min at 37°C. Cells were filtered through 100-µm cell strainers, washed in isolation buffer, and stained in PBS supplemented with 0.25% BSA, 2 mM EDTA, and 0.1% sodium azide. Antibodies used included anti-CD45, anti-Foxp3, anti–IL-18Rα, anti-ST2, anti-CD62L, anti-CD103, anti–PD-1, anti-GITR, anti–CTLA-4, anti-KLRG1, anti-Ki67, anti-CD5, anti-NK1.1, anti-CD45R (B220), anti–IL-17, anti–IL-4, anti-IFNγ, and anti–Ly-6C (eBioscience); anti-TCRβ, anti-CD3, anti-CD4, anti-CD8, anti-CD127, anti-CD11B, anti-MHCII, and anti-Gr1 (BioLegend); anti-CD44 and anti-CD25 (Tonbo); anti–IL-5 and anti-TNFα (BD PharMingen); and anti-AREG (R&D Systems). For exclusion of dead cells, samples were first stained with Ghost Dye (Tonbo) cell viability reagent. Intracellular staining for cytokines, Foxp3, AREG, Ki-67, and CTLA-4 was performed using the Foxp3/transcription factor staining buffer set (eBioscience) as per manufacturer’s protocol. The H2-Kb OVA257–264 tetramer was obtained from the NIH Tetramer Core Facility.
3
Characterization of Chronic Myeloid Leukemia Cells
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Chronic myeloid leukaemia cell line, LAMA84, was obtained from DSMZ (Braunschweig, Germany); human primary CD34+ cells and bone marrow primary stromal cells (BMSCs) were obtained from Lonza (Basel, Switzerland). LAMA84 cells were cultured in RPMI 1640 medium, supplemented with 10% foetal bovine serum (FBS), 2 mM L‐glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Euroclone, UK). CD34+ cells were cultured in IMDM medium, supplemented with 15% FBS, 2 mM L‐glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Euroclone, UK). BMSCs were cultured in MyeloCult H5100 (STEMCELL Technologies Inc., Vancouver, BC, Canada). Gefitinib or Erlotinib (Cayman Chemical, Ann Arbor, MI, USA) was solubilized at 10‐mM stock solution in DMSO and stored at −20°C. Neutralizing antibody anti‐AREG (R&D Systems, Abingdon, UK) was reconstituted at 0.2 mg/ml in sterile PBS, aliquoted and stored at −20°C. Recombinant Areg (R&D Systems, Abingdon, UK) was reconstituted at 0.1 mg/ml in sterile PBS, aliquoted and stored at −20°C. Working dilutions, where necessary, were prepared in medium. All other reagents were purchased from Sigma‐Aldrich (St. Louis, MO, USA), if not cited otherwise.
4
Tumor Modeling and Immunotherapy Protocols
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MC38 cell line was generously provided by Z. Guo (University of Pittsburgh, School of Medicine) and cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P.S). Generation of B16–IL-33 and B16-vec tumor cell lines was previously reported (10 (link)). All cell lines were tested for mycoplasma routinely and with negative results. B16 and B16–IL-33 cells were cultured in RPMI 1640 medium with 10% FBS and 1% P.S. One million MC38 cells were injected intradermally into the right flank of the mice. For B16 models, 0.1 million cells were injected intradermally. IL-33 protein (10 μg), anti–PD-1 (200 μg per mouse; Bio X Cell, clone: J43), or anti-Areg (50 μg per mouse; R&D Systems, clone: 206220) was injected intraperitoneally on the fifth day after tumor inoculation, for a total of four times with 4-day intervals.
5
Exosomes Modulate Cell Signaling in CML
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HS5 cells were starved for 2 h in a serum‐free medium and treated or not for different times (30 min., 3 h, 6 h, 24 h) with LAMA84− exosomes (20–50 μg/ml) ± gefitinib 30 μM or erlotinib 30 μM. Neutralizing antibody anti‐AREG (20 ng/ml) was incubated with LAMA84 exosomes (50 μg/ml) for 2 h at 37°C and then used to treat HS5 for 3 h. Bone marrow primary stromal cells were treated or not for different times (30 min., 18 h) with LAMA84− exosomes (50 μg/ml) ± gefitinib 30 μM. Total protein lysates were obtained at the end of each treatment. Protein lysates from LAMA84 cells, from CML patients cells, from HS5, from BMSC, from exosomes or from conditioned medium of LAMA84 cells were analysed by SDS‐PAGE in reducing conditions followed by Western blotting as previously described 6 . Antibodies used in the experiments were as follows: anti‐EGFR, pEGFR, annexin A2 and β‐actin (all from Cell Signalling Technology, Lane Danvers, MA, USA); anti‐SNAIL and MMP9 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti‐AREG (R&D systems, Abingdon, UK).
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