Following Didox treatment and IL-33 activation for 18 hours, cytokines were measured in the cell culture supernatant via ELISA. Murine ELISA kits were purchased from Biolegend for IL-6, TNF, and MCP-1 (CCL2) and Peprotech (Rocky Hill, NJ) for IL-13 and MIP-1α (CCL3). Assays were performed in duplicate according the manufacturers’ protocols.
Ccl2 mcp 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CCL2/MCP-1 is a laboratory tool used for the detection and quantification of the chemokine Monocyte Chemoattractant Protein-1 (MCP-1) in various biological samples. It is a key component in the assessment of inflammatory processes and immune system function.
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Lab products found in correlation
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Recombinant human cxcl10, by BioLegend (1 mentions)
12 protocols using ccl2 mcp 1
1
Cytokine Profiling after Didox and IL-33
2
Cell Competition Assay with Signaling Modulators
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The following reagents and recombinant proteins were used for cell competition assay: sodium dichloroacetate (Sigma-Aldrich, #347795); recombinant human IL-6 (PEPRO TECH, #200-06), IL-8 (PEPRO TECH, #200-08M), GDF-15/IMC-1 (PEPRO TECH, #120-28C), MCP-1 (CCL2, PEPRO TECH, #300-04), HGF (PEPRO TECH, #100-39), TGF-β1 (PEPRO TECH, #100-21), CD105 protein (Abcam, #ab54338), BSG protein (P01, Abnova, #H00000682-P01), DPP4 protein (P01, Abnova, #H00001803-P01), pentraxin 3/TSG-14 (R&D Systems, #1826-TS), and uPAR protein (84–95) (scrambled peptide, Anygen, #C180084).
3
Cytokine-Mediated Podocyte Responses
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Primary human GECs, primary podocytes subcultured from human donor kidney, and organoid-derived podocytes were treated with 50 ng/mL, 20 ng/mL, or 10 ng/mL concentration of the following cytokines: recombinant human CXCL9 (BioLegend, 578102); recombinant human CXCL10 (BioLegend, 573502); recombinant human CXCL13 (BioLegend, 573502); IL-1β (PeproTech, 200-01B); IL-15 (PeproTech, 200-15); IL-18 (BioLegend, 592102); IL-8 (BioLegend, 574202); IFN-α1 (MilliporeSigma, SRP4596); IFN-β (PeproTech, 300-02BC); IFN-γ (MilliporeSigma, I17001); IL-6 (PeproTech, 200-06); TNF-α (PeproTech, 300-01A); MCP-1 (CCL2; PeproTech, 300-04); MIP-1α (CCL3; PeproTech, 300-08); IL-10 (PeproTech, 200-10); IL-7 (PeproTech, 200-07); IL-2 (PeproTech, 200-02); and G-CSF (PeproTech, 300-23). A standardized cytokine concentration of 50 ng/mL was predetermined on the basis of precedent from human cell treatments evaluating cytokine-shock syndromes in COVID-19 infection (17 (link)). Follow-up experiments used concentrations of 20 ng/mL and 10 ng/mL. Subsequent treatments, including organoid and organoid-derived podocyte experiments, were performed using a cytokine concentration of 10 ng/mL. The JAK1/2 inhibitor baricitinib (INCB028050; Selleckchem, S2851) was used at 10 μM final concentration in all experiments.
4
Quantitative Real-Time PCR for Kidney Injury Markers
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Reverse transcription was performed on equal amounts of total RNA by using random hexanucleotide primers to produce a cDNA library for each sample. Real-time PCR reactions were run on an iCycler iQ Real-Time PCR Detection System using Taqman Universal PCR Master Mix (Applied Biosystems, P/N 4304437). KIM-1, osteopontin (OPN)/secreted phosphoprotein-1, fibronectin-1, monocyte chemotactic protein-1 (MCP-1/CCL2), and β-actin gene-specific Taqman probe and primer sets were obtained from Applied Biosystems as Assays-on-Demand (AOD) gene expression products. The AOD identification numbers were Rn00597703_m1 for KIM-1, Rn00563571_m1 for OPN, Rn00569575_m1 for fibronectin-1, Rn00580555_m1 for MCP-1, and 4331182 for the rat β-actin endogenous control. Each sample was run in triplicate, and the comparative threshold cycle (Ct) method was used to quantify fold increase (2–∆∆Ct) compared with controls.
5
Quantifying MCP-1 Expression in LPS-Stimulated THP-1 Cells
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From the LPS-stimulated THP-1cells, total RNA was isolated using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA), and 1 µg of total RNA was used to generate complementary DNA (cDNA) using AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Real time semi-quantitative polymerase chain reaction (PCR) was carried out using TaqMan® Fast Universal PCR Master Mix (2×) (Applied Biosystems, Foster City, CA, USA) and ViiA7 Real-Time PCR Detection System (Applied Biosystems). Human TaqMan probes and primers were purchased from Applied Biosystems: MCP-1/CCL2 (assay ID: Hs00234140_m) [18 (link)].
6
Inflammatory Mediator Procurement Protocol
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Lipopolysaccharide (O111:B4; LPS) was purchased from Sigma‐Aldrich (Schnelldorf, Germany). Recombinant murine IL‐6, CCL2 (MCP‐1) and LIF were from PeproTech (London, UK). Prostaglandin E2 and celecoxib were purchased from R&D Systems (Abingdon, UK).
7
Quantification of Plasma Cytokine and Chemokine Levels
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Plasma cytokine and chemokine levels of the following six molecules were measured using ELISA kits, in accordance with the manufacturer’s instructions: interferon gamma (IFN-γ) (cat. 900-K27, Peprotech, New Jersey, NJ, USA); tumor necrosis factor alpha (TNF-α) (cat. 900-K25, Peprotech); C-C motif chemokine ligand 2 (CCL2/ MCP-1) (cat. 900-K31, Peprotech); C-X-C motif chemokine ligand 10/ interferon protein-10 (CXCL10/IP-10) (cat. 900-K39, Peprotech); interleukin-6 (IL-6) (cat. DY206, R&D Systems, Minneapolis, MN, USA); and IL-10 (cat. DY217B, R&D Systems). Standard curves of known concentrations of recombinant human cytokines or chemokines were used to convert optical density (OD) into concentration units (pg/mL). The plates were read in a SpectraMax Paradigm® machine (Molecular Devices, CA, USA).
8
Carvedilol's effect on T. cruzi infection
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To analyze the interference of carvedilol therapy on inflammatory response associated with T. cruzi experimental infection, immunoassays were performed for inflammatory cytokine and chemokine TNF and CCL2/MCP-1 (PeproTech, NJ, USA), respectively, and for the regulatory cytokine IL-10 (PeproTech, NJ, USA). Blood from the orbital venous sinus (0.5 mL) was collected during euthanasia and centrifuged (1500 g for 15 minutes at 4°C). The plasma was stored at −80°C. Next, these samples were used to measure TNF, CCL2, and IL-10, according to the protocol recommended by the manufacturer. The samples were simultaneously measured in triplicate.
9
Standardized T-cell Migration Assay
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Migration T-cell assays were performed based on previous standardization assays and conditions reported for T-cell migration (17 (link)). Chemoattractants CCL2 (MCP-1) (200 ng/ml) and CCL19 (200 ng/ml) (PeproTech Cranbury, NJ; USA) in 150 μl of RPMI media were added alone or together to the bottom well of a 96-well chemotaxis plate (Cytoselect 96-well cell migration assay-fluorometric format, Cell Biolabs, Inc. San Diego, CA, USA.). T cells cultured 24 h with supernatants were recovered and suspended in RPMI 1640 serum-free media (4 × 106 cells/ml), and 100 μl of suspension cells were added on top of the membrane (5 μm pore size). Cells were allowed to migrate for 3 h at 37°C in 5% CO2. Cells in the membrane were recovered using 150 μl of cell detachment solution and lysed. The total number of transmigrated cells was quantified using CyQuant® GR Dye (Synergy™ HT, BioTek, Inc., USA). Cells in RPMI 1640 with BSA 1% were used as blanks for each chemokine.
10
Quantifying Plasma Cytokine Profiles
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The concentrations of 9 parameters, including cytokines and chemokines present in the plasma samples, were estimated using ELISA kits, in accordance with the manufacturer’s instructions. The cytokines/chemokines that were assayed included IFN-γ (cat. 900-K27, Peprotech, NJ, USA); TNF-α (cat. 900-K25, Peprotech, NJ, USA); CCL2/ MCP-1 (cat. 900-K31, Peprotech, NJ, USA); CXCL10/IP-10 (cat. 900-K39, Peprotech, NJ, USA); IL-6 (cat. DY206, R&D Systems, MN, USA); IL-10 (cat. DY217B, R&D Systems, MN, USA); CCL5/RANTES (cat. 900-K33, Peprotech, NJ, USA); CCL4/ MIP1-β (cat. 900-T36, Peprotech, NJ, USA); and CXCL-8/ IL-8/ (DY208, R&D Systems, MN, USA). Standard curves of known concentrations of recombinant human cytokines or chemokines were used to convert optical density (OD) into concentration units (pg/mL). The levels of cytokines/chemokines were analyzed using a SpectraMax Paradigm® instrument (Molecular Devices, San Jose, CA, USA).
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