BC cell line MCF-7 was grown in MEM medium (SH30024.01B; Hyclone) supplemented with 10% FBS (10099-141; Gibco) in a humidified atmosphere with 5% CO2 at 37°C. Three different small interfering RNAs (siRNAs) targeting ARF6 (see Table S1 ), miR-760 mimics, inhibitor, and corresponding controls were synthesized by RiboBio (Guangzhou, China). For chemokine treatment, MCF-7 cells were exposed to 20 ng/mL CCL18 or CCL20 (both from R&D, Shanghai, China) for 1 h. For ARF6 function, MCF-7 cells were transfected with si-ARF6-1, si-ARF6-2, si-ARF6-3, or si-NC, followed by CCL18 treatment. For Src/PI3K/Akt signaling, CCL18-treated MCF-7 cells were treated with Src activator MCB-613 (8 μΜ), PI3K activator 740 Y-P (10 μΜ), or SC for 2 h, followed by si-ARF6-2 transfection. For miR-760 function, MCF-7 cells were transfected with miR-760 mimics or inhibitor. All transfection protocols were conducted according to the instructions provided by Lipofectamine 3000 (Invitrogen, USA) for 48 h.
Ccl18
Manufactured by R&D Systems
Sourced in United States, China
CCL18 is a recombinant human protein that functions as a chemokine. Chemokines are a family of small cytokines that are involved in the inflammatory response and immune cell recruitment.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Il 17a, by R&D Systems (2 mentions)
Ccl20, by R&D Systems (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Il 1ra, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Myeloperoxidase mpo, by R&D Systems (1 mentions)
C reactive protein (crp), by R&D Systems (1 mentions)
Periostin, by R&D Systems (1 mentions)
Il 17f, by Thermo Fisher Scientific (1 mentions)
Il 22, by R&D Systems (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Ifn γ, by BD (1 mentions)
Il 13, by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
Cxcl12, by Thermo Fisher Scientific (1 mentions)
Dcl180b, by R&D Systems (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Ccl22, by Thermo Fisher Scientific (1 mentions)
Dta00d, by R&D Systems (1 mentions)
10 protocols using ccl18
1
Modulating ARF6 and miR-760 in Breast Cancer
2
Evaluating Antioxidant and Anti-inflammatory Effects of myo-Inositol
3
Cytokine Measurement by ELISA
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Cytokines were measured by ELISA according to the manufacturer’s recommendations. IL-17A, IL-22, CCL20, IL-13, IL-5, IL-10, TGF-β1, periostin and CCL18 kits were from R&D systems (Minneapolis, MN, USA), IL-17F from eBiosciences and IFN-γ from BD Biosciences (Franklin Lakes, NJ, USA). The lower detection limit was 15.6 pg/ml for IL-17A, IL-17F and CCL20, 31.2 pg/ml for IL-22, IL-5, IFN-γ, and TGF-β, 93.8 pg/ml for IL-13, 375 pg/ml for periostin and 7.8 pg/ml for CCL18.
4
Chemotaxis Assay for Natural Killer Cells
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Chemotaxis experiments were performed in a Boyden chamber as previously described [31 (link)]. NK cells were incubated for 2 h with CCL18 (at a concentration of 10–10 to 10–7 M, R&D systems), or CXCL12 (10−7 M, Peprotech Inc, Rocky Hill, NJ, USA) and complete RPMI as positive and negative controls, respectively. Each condition was performed in triplicate. NK cells were counted in the inferior chamber using a hemocytometer. Results are expressed as chemotactic index = (migration towards the tested chemokine)/(migration obtained with complete RPMI).
To phenotype NK cells migrating in response to CCL18, chemotaxis experiments were performed using Transwells (5 µm, 24 wells, Corning). CCL18 (5 × 10−8 M) was added to the lower chamber in a 500 µL volume. A total of 5 × 105 NK cells/100 µL were added to the upper chamber and incubated for 2 h at 37 °C in 5% CO2. Migrating NK cells were obtained from the lower chamber and stained for phenotype analysis. NK cell phenotype was also assessed before migration. Results are expressed as percentage of migrating NK cells = (migrating NK cell number)/(NK cell number before migration) × 100, or enrichment after migration = (% of NK cells positive for the marker after migration)/(% of NK cells positive for the marker before migration).
To phenotype NK cells migrating in response to CCL18, chemotaxis experiments were performed using Transwells (5 µm, 24 wells, Corning). CCL18 (5 × 10−8 M) was added to the lower chamber in a 500 µL volume. A total of 5 × 105 NK cells/100 µL were added to the upper chamber and incubated for 2 h at 37 °C in 5% CO2. Migrating NK cells were obtained from the lower chamber and stained for phenotype analysis. NK cell phenotype was also assessed before migration. Results are expressed as percentage of migrating NK cells = (migrating NK cell number)/(NK cell number before migration) × 100, or enrichment after migration = (% of NK cells positive for the marker after migration)/(% of NK cells positive for the marker before migration).
5
Cytokine Secretion in Breast Cancer
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Fresh breast cancer puncture specimens or cells exposed to the indicated treatment were cultured in DMEM supplemented with 10% FBS for 24 h, and the supernatant was collected by centrifugation for subsequent ELISA analysis. IL-6 (eBioscience Cat# 88-7066-86), IL-8 (eBioscience Cat# 88-8086-86) and CCL18 (R&D Systems Cat #DCL180B) ELISA kits were used according to the manufacturers’ instructions.
6
Multiplex Biomarker ELISA Assay
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Human CCL2 (R & D Systems # DCP00), CCL13 (Thermo Scientific #), CCL17 (Thermo Scientific # EHCCL17), CCL18 (R & D Systems), CCL21 (R & D Systems # DY366), CCL22 (Thermo Scientific #), CCL26 (Raybiotech # ELH-Eotaxin3), IL1B (Invitrogen # KHC00012), IL1RN (R & D Systems # DRA00B), and TNF (R & D Systems # DTA00D) were measured in cell culture supernatants by ELISA, according to the manufacturer's instructions. Results were obtained using a Spectramax 96 well Spectrophotometer.
7
Multiplex ELISA Profiling of Secreted Proteins
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Secreted proteins, including TNF-α, IL-1β, IL-2, TGF-β, GM-CSF, IL-17A, TREM2, serpin E1/PAI-1, G-CSF, CCL18, and MMP-8 (all R&D systems, Minneapolis, MN, USA), were measured in the Nas or CAAs supernatants using sandwich ELISA in accordance with the methods provided by the manufacturer’s instructions. The detected signals were visualized using a microarray laser scanning system (GenePix, USA).
8
Quantification of Inflammatory Cytokines
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Enzyme-linked immunosorbent assays (ELISAs) were performed according to manufacturer's instructions to quantify the concentrations of CCL18, IL-1ra, IL-6, and TNFα (R&D Systems, Minneapolis, MN, USA) released in the cell culture supernatants. These selected cytokines were chosen based on our previous research in which CCL18, IL-1ra, IL-6, and TNFα were the most discriminative for the different macrophages phenotypes [18, 20] . All measurements fitted within the standard curve, for every material and cytokine different dilutions had to be made of the culture medium, ranging from a 3 to 100 times dilution. C-Reactive Protein (CRP) levels in the plasma were determined using the standard technique at the hospital's laboratory (Dimension Vista® System, Flex reagent cartridge, Siemens Healthcare Diagnostics Products, Germany) and expressed in mg/L. CRP is a very common used parameter in all hospitals to detect early systemic inflammation, also prior to surgery, especially in obese patients.
9
Macrophage Polarization Characterization
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To confirm macrophage polarization, IL-6, CCL18 and sCD163 were measured in the second MCM batch. In our previous work, we have characterized M(IFNγ+TNFα), M(IL-4) and M(IL-10) primary human monocyte-derived macrophages based on gene expression and protein production [3, 8, 10, 11] . IL-6 was found to be a good marker for M(IFNγ+TNFα), CCL18 for M(IL-4) and soluble CD163 (sCD163) for M(IL-10) which was also supported by others [12, 13] . IL-6 (PeproTech), CCL18 (R&D Systems) and sCD163 (PeproTech) protein concentrations were quantified in the MCM using enzyme-linked immunosorbent assays (ELISAs) according to manufacturer's instructions. To check for possible nutrient depletion of the conditioned medium, glucose was measured. Glucose concentration was 0.83 g/L for M(IFNγ+TNFα) MCM, 0.93 g/L for M(IL-4) MCM, 0.92 g/L for M(IL-10) MCM and 1.0 g/L in non-conditioned medium. Since MCM was mixed 1:1 with fresh medium, the difference in glucose between the conditions was maximally 5.5% and considered negligible.
10
Evaluating Protein Adsorption on Materials
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To evaluate potential adsorption of the proteins by the materials, the materials were pre-conditoned for 2 hours in non-heat inactivated FCS with agitation, followed by 2 hours incubation in X-VIVO/20% FCS in a tube rotator at 37°C. Next, the materials were transferred to well plates and incubated in X-VIVO/20% FCS for 2 days. After this period, the materials were transferred to new well plates and incubated in medium containing either 1 ng/mL IL-6 (Peprotech), 250 pg/mL CCL18 (R&D Systems), 1.25 ng/mL IL 1RA (R&D Systems), 500 pg/mL TNFα or no cytokine. After an additional incubation day, the media were harvested, centrifuged at 300 x g and stored at -80°C until cytokine quantification. The use dosages were based on the detection ranges of the enzyme-linked immunosorbent assays (ELISAs) that were used to determine cytokine concentrations.
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