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Ccl5 rantes

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CCL5/RANTES is a lab equipment product designed for the detection and quantification of the CCL5 (C-C Motif Chemokine Ligand 5) protein, also known as RANTES (Regulated on Activation, Normal T Expressed and Secreted). It is a tool used for research and analytical purposes in various fields, including immunology, cell biology, and biochemistry.

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Lab products found in correlation

Ccl2 mcp 1, by Thermo Fisher Scientific (2 mentions) Tnf α, by Thermo Fisher Scientific (2 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Mmp 9, by Thermo Fisher Scientific (2 mentions) Cxcl8 il 8, by Thermo Fisher Scientific (2 mentions) Hydrogen peroxide, by Thermo Fisher Scientific (1 mentions) Euk 134, by Cayman Chemical (1 mentions) Rat tail collagen 1, by BD (1 mentions) Raw 264.7 macrophage cells, by American Type Culture Collection (1 mentions) β actin antibody a5441, by Merck Group (1 mentions) Collagenase 1, by Merck Group (1 mentions) Sigenome smartpool, by Thermo Fisher Scientific (1 mentions) Cellsens dimensions software, by Olympus (1 mentions) Siport neofx transfection agent, by Thermo Fisher Scientific (1 mentions) Matrigel, by BD (1 mentions) Sdf 1, by Thermo Fisher Scientific (1 mentions) Transwell chamber plate, by Corning (1 mentions) Mip 1α, by Thermo Fisher Scientific (1 mentions) Transwell system, by Corning (1 mentions) Facscan, by BD (1 mentions)

9 protocols using ccl5 rantes

1

Isolation and Culture of Raw 264.7 Macrophages

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The raw 264.7 macrophage cells (ATCC, Manassas, VA, USA) were maintained in DMEM (high glucose) media containing 10% FBS and 100 U/mL penicillin/streptomycin in a 37 °C incubator supplemented with 5% CO2. To obtain the conditioned media, 3 × 106 cells per 10 cm dish were grown in media used for acinar cell 3D collagen explant culture (Waymouth’s media plus supplements, see below) and supernatants were collected. The conditioned media was freshly prepared for each experiment. The PKD1 antibody (AP06569PU-N) was from Acris (San Diego, CA, USA), the β-actin (A5441) antibody was from Sigma-Aldrich (St. Louis, MO, USA). The secondary HRP-linked anti-mouse or anti-rabbit antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). The recombinant murine CCL5/RANTES and TNFα were purchased from PeproTech (Rocky Hill, NJ, USA). The recombinant TGFα was from R&D Systems (Minneapolis, MN, USA). The hydrogen peroxide was from Thermo Fisher (Rochester, MN, USA) and the EUK134 was from Cayman Chemical (Ann Arbor, MI, USA). The PKD-specific inhibitor kb-NB-142-70 was described previously [14 (link),20 (link)]. The collagenase I was from Millipore/Sigma (St. Louis, MO, USA). The rat tail collagen I was from BD Biosciences (San Diego, CA, USA).
Döppler H.R., Liou G.Y, & Storz P. (2022). Generation of Hydrogen Peroxide and Downstream Protein Kinase D1 Signaling Is a Common Feature of Inducers of Pancreatic Acinar-to-Ductal Metaplasia. Antioxidants, 11(1), 137.
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2

Endothelial Cell Tube Formation and Migration

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For tube formation endothelial cells were grown on Matrigel™ (BD Biosciences, following standard methods [27 (link)]. Tube number and length were analyzed from randomly selected fields. For wound healing a scratch (wound) was made across a cell monolayer using a sterile tip. Image analysis was conducted using ImageJ software. Relative clearance rate was determined using the equation: (distance=0h – distance=24h)/ distance=0h × 100 % [27 (link)]. For migration assays, we used a transwell 8 μm polycarbonate membrane assay (Corning, Tewksbury, MA), with either conditioned media, or recombinant purified CCL5/RANTES (10 nM) (PreproTech, Rocky Hill, NJ) as chemoattractant. After four hours membranes were imaged using the IX71 fluorescent microscope and counted using CellSens Dimensions software (Olympus, Tokyo Japan).
Endothelial cells were transfected with siRNAs (0.04 pmol/μl) using siPORT NeoFX Transfection Agent (Ambion, Carlsbad, CA) [27 (link)], and/or treated with the small molecular inhibitor of CCR5, maraviroc (100 nM). Pooled siRNAs specifically designed to inhibit mouse or human CCR5 (siGENOME SMARTpool) and Cyanine 3 (Cy3) labeled siGLO RISC-Free Control siRNA were obtained from Thermo Scientific (Waltham, MA).
Sax M.J., Gasch C., Athota V.R., Freeman R., Rasighaemi P., Westcott D.E., Day C.J., Nikolic I., Elsworth B., Wei M., Rogers K., Swarbrick A., Mittal V., Pouliot N, & Mellick A.S. (2016). Cancer cell CCL5 mediates bone marrow independent angiogenesis in breast cancer. Oncotarget, 7(51), 85437-85449.
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3

Macrophage Migration Assay Using Transwell

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In vitro cell migration was assayed using 24-well Transwell chamber plates (pore size: 5 μm, Corning). The upper well, containing 106 macrophages, was incubated at 37°C for 4 hours. The lower chamber contained RPMI with and without SDF1 (60 ng/ml), MIP-1α (50 ng/ml), or CCL5 (RANTES) (50 ng/ml) (PeproTech). Cells were allowed to migrate through the membrane into the lower chamber. Macrophages migrating to the bottom wells were counted on a FACSCalibur flow cytometer. The chemotaxis index was calculated from the ratio of migrated macrophages in the presence and absence of the respective chemokine (38 (link)).
Aflaki E., Stubblefield B.K., Maniwang E., Lopez G., Moaven N., Goldin E., Marugan J., Patnaik S., Dutra A., Southall N., Zheng W., Tayebi N, & Sidransky E. (2014). Macrophage Models of Gaucher Disease for Evaluating Disease Pathogenesis and Candidate Drugs. Science translational medicine, 6(240), 240ra73.
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4

T cell Chemotaxis Assay for Melanoma

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Chemotaxis assay for T cells migration was evaluated using a 48-well Transwell system (5 μm pore size, Corning Costar, Corning, NY, USA) with T cells placed in the upper chamber (1 × 106 cells/mL, 100 μL). Assay medium (RPMI 1640 containing 2% FBS), CCL5/RANTES (20 ng/mL, PeproTech, Rocky Hill, NJ, USA) chemotactic for T cells (a positive control), conditioned medium from control SC and SC-treated with tumor-conditioned medium (B16, Ret and BPWT melanomas) were added to the bottom chamber (600 μL). Migration of T cells was assessed after 4-h incubation at 37 °C, 5% CO2. Cells transmigrated thought the membrane were collected and acquired on FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) for 1 min for enumeration. Data are reported as the mean numbers of transmigrated cells from triplicate wells.
Shurin G.V., Vats K., Kruglov O., Bunimovich Y.L, & Shurin M.R. (2022). Tumor-Induced T Cell Polarization by Schwann Cells. Cells, 11(22), 3541.
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5

Splenic T Cell Stimulation Assay

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Cell suspensions from the spleen of B6-Il10-/- mice were made and erythrocytes were lysed as described previously (23 (link)). Splenic T cells were isolated by negative selection using magnetic cell separation. Therefore, cell suspension was incubated with anti-MHCII (BD Biosciences) and IgG beads (MACS, Miltenyi) each for 20 min. Isolated cells were stained with CFSE (5 mmol) for 2 min and washed with MACS buffer. Next, 2x106 cells/well were cultured with or without anti-CD3 antibody (Biolegend) for 48 hours in RPMI medium (Biochrom, Berlin, Germany) with 10% FCS (GE Healthcare Life Sciences, Buckinghamshire, UK), 1% Pen/Strep (Gibco, Thermo Fisher Scientific, Waltham, USA), 0.3 mg/ml Glutamin (Biochrom), and 10 µM β-mercaptoethanol containing recombinant protein (770 ng/mL CCL5/RANTES 134 ng/mL CCL2, 200 ng/mL CCL7, and 150 ng/mL CXCL16; all Peprotech) for stimulation. Stimulation experiments were performed in duplex and data were generated from 2–5 independent experiments.
Basic M., Peppermüller P.P., Bolsega S., Bleich A., Bornemann M., Bode U, & Buettner M. (2021). Lymph Node Stromal Cells From Different Draining Areas Distinctly Regulate the Development of Chronic Intestinal Inflammation. Frontiers in Immunology, 11, 549473.
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6

Plasma Cytokine Profiling in COVID-19

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Blood samples were collected from the controls (n = 9) and all COVID-19 patients (n = 66) from the antecubital vein into 4 mL tubes with EDTA as anti-coagulant at the earliest time-point after hospital admission (T1). Additional blood samples were collected from 14 survivors and 12 non-survivors COVID-19 patients at the discharge time from the hospital (T2: 0 to 72 h before leaving hospital or death). Plasma samples were obtained by centrifugation (2000g, 10 min), aliquoted and immediately kept at −80 °C until analysis.
Plasma levels of IL-6, IL-10, TNF-α, TGF-β1 (all from Invitrogen Life Sciences, USA), IFN-γ, CCL2/MCP-1, CCL4/MIP-1β, and CCL5/RANTES (all from Peprotech, USA) were analyzed by Enzyme Linked Immunosorbent Assay (ELISA) following the manufacturer’s procedure using a microplate reader (EzBiochrom, USA). The intra-assay coefficient of variability was <7.5%. The detection limits of each cytokine were: IL-6, 2–200 pg/mL; IL-10, 4–200 pg/mL; IFN-γ, 10–300 pg/mL; TNF-α, 2–200 pg/mL; TGF-β1, 2–500 pg/mL; CCL5/RANTES, 20–1000 pg/mL; CCL4/MIP-1β, 50–1500 pg/mL; CCL2/MCP-1, 50–1000 pg/mL. The soluble form of CD14 was analyzed using Human CD14 ELISA Kit from Invitrogen (USA), with detection limits ranging 8.23–8000 ng/mL.
Teixeira P.C., Dorneles G.P., Santana Filho P.C., da Silva I.M., Schipper L.L., Postiga I.A., Neves C.A., Rodrigues Junior L.C., Peres A., Souto J.T., Fonseca S.G., Eller S., Oliveira T.F., Rotta L.N., Thompson C.E, & Romão P.R. (2021). Increased LPS levels coexist with systemic inflammation and result in monocyte activation in severe COVID-19 patients. International Immunopharmacology, 100, 108125.
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7

Quantification of Inflammatory Mediators in Dengue

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The quantification of chemokines and cytokines was performed using serum from patients with a confirmed diagnosis of dengue. ELISA assay was used to quantify CX3CL1/Fractalcina (catalog # DCX310, R&D Systems, Minneapolis, MN, USA), CXCL10/IP-10 (catalog # 900-T39, Peprotech, Rocky Hill, NJ, USA), CCL2/MCP-1 (catalog # 900-T31, Peprotech, Rocky Hill, NJ, USA), CCL5/RANTES (catalog # 900-T33, Peprotech, Rocky Hill, NJ, USA), and MMP-9 (catalog # KHC3061, Invitrogen, Carlsbad, CA, USA).
Cipitelli M.D., Paiva I.A., Badolato-Corrêa J., Marinho C.F., Fiestas Solórzano V.E., da Costa Faria N.R., de Azeredo E.L., de Souza L.J., da Cunha R.V, & de-Oliveira-Pinto L.M. (2022). Subsets of Cytokines and Chemokines from DENV-4-Infected Patients Could Regulate the Endothelial Integrity of Cultured Microvascular Endothelial Cells. Pathogens, 11(5), 509.
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8

Multiplex Biomarker Quantification in Plasma

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ANG-2 (Boster Biological Tecnhology Co., LTD, Pleasanton, CA, USA), MMP-9 (ThermoFisher, Monza, Italy), CCL5/RANTES (ThermoFisher, Monza, Italy), VEGF-A (Boster Biological Tecnhology Co., LTD, Pleasanton, CA, USA), IL-8/CXCL8 (ThermoFisher, Monza, Italy), and IL-6 (ThermoFisher, Monza, Italy) concentrations were assessed using highly sensitive enzyme-linked immunosorbent assay kit in triplicate on plasma samples. Enzyme-linked immunosorbent assay was performed according to the manufacturer’s instructions.
Dei Cas M., Carrozzini T., Pollaci G., Potenza A., Nava S., Canavero I., Tinelli F., Gorla G., Vetrano I.G., Acerbi F., Ferroli P., Ciceri E.F., Esposito S., Saletti V., Ciusani E., Zulueta A., Paroni R., Parati E.A., Ghidoni R., Bersano A, & Gatti L. (2021). Plasma Lipid Profiling Contributes to Untangle the Complexity of Moyamoya Arteriopathy. International Journal of Molecular Sciences, 22(24), 13410.
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9

Quantification of Secreted Angiogenic Factors

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VEGF-A (PEPROTECH, Rocky Hill, NJ, USA), HGF (R&D Systems, Minneapolis, MN, USA), TGF-β1 (R&D Systems, Minneapolis, MN, USA), IL-8/CXCL8 (ThermoFisher, Monza, Italy), CCL2/MCP-1 (Diaclone, Besancon, France), and CCL5/RANTES (ThermoFisher, Monza, Italy) concentrations were assessed using highly sensitive enzyme-linked immunosorbent assay kit in triplicate samples obtained from EPC conditioned media. Enzyme-linked immunosorbent assay was performed according to the manufacturer’s instructions.
Tinelli F., Nava S., Arioli F., Bedini G., Scelzo E., Lisini D., Faragò G., Gioppo A., Ciceri E.F., Acerbi F., Ferroli P., Vetrano I.G., Esposito S., Saletti V., Pantaleoni C., Zibordi F., Nardocci N., Zedde M.L., Pezzini A., Di Lazzaro V., Capone F., Dell’Acqua M.L., Vajkoczy P., Tournier-Lasserve E., Parati E.A., Bersano A, & Gatti L. (2020). Vascular Remodeling in Moyamoya Angiopathy: From Peripheral Blood Mononuclear Cells to Endothelial Cells. International Journal of Molecular Sciences, 21(16), 5763.
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