Size-matched polyp and normal tissues were collected from the small intestine of B6.ApcMin/+ mice and B6.Apc+/+ mice, respectively. Tissues were frozen in Qiagen RLT buffer (Valencia, CA) and RNA extracted with Qiagen RNeasy Mini Kits. Quantitative RT-PCR was done with SYBR Green (Quanta BioSciences, Gaithersburg, MD) to measure the expression levels of F4/80, IL-6, IL-1b, TNF and Myc in adipose or intestinal tissues (see Supplementary Table 2 for primer sequences). Tissues for Western blot analysis were immediately submerged in RIPA buffer supplemented with phosphatase and protease inhibitors, frozen in liquid nitrogen, and stored at −80°C. All antibodies (AKT, pAKT, IKK, pIKK, NFκB, p65 NFκB, STAT3, pSTAT3) were purchased from Cell Signaling Technology (Beverly, MA) and used according to manufacturer’s instructions. Homogenates from intestinal samples were used to measure intestinal inflammation using R&D Systems (Minneapolis, MN) IL-1β, IL-10, TNF-α, VEGF, and IL-23 ELISAs and the Milliplex Map Mouse Cytokine/Chemokine Panel from EMD Millipore (Temecula, CA).
Milliplex map mouse cytokine chemokine panel
Manufactured by Merck Group
Sourced in United States
The MILLIPLEX MAP Mouse Cytokine/Chemokine Panel is a multiplex assay kit designed to detect and measure multiple mouse cytokines and chemokines simultaneously in a single sample. The panel uses Luminex technology to quantify the levels of these analytes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex system, by Bio-Rad (7 mentions)
Bio plex 200, by Bio-Rad (3 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Vascular endothelial growth factor (vegf), by Merck Group (1 mentions)
Il 1β, by Merck Group (1 mentions)
Il 10, by Merck Group (1 mentions)
P ikk, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Tnf α, by Merck Group (1 mentions)
Sybr green, by Quanta Biosciences (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Pierce protease inhibitor tablet, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
Hsp70, by R&D Systems (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
21 protocols using milliplex map mouse cytokine chemokine panel
1
Intestinal Inflammation Profiling in ApcMin Mice
2
Tumor Interstitial Fluid and Serum Profiling
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To obtain TIF, 0.1–0.3 g of fresh tumor tissues were cut into small pieces (1–3 mm3), processed in homogenizer for 30–60 s and then placed in a 15-mL conical plastic tube containing cold PBS (1 mL PBS per 0.25 g tumor tissue). Samples were centrifuged at 100 x g for 3 min and the supernatants were transferred to new microtubes. Samples were further centrifuged at 2500 × g for 20 min at 4 °C. To obtain serum, mice were anesthetized with a ketamine and xylazine cocktail (100 mg/kg and 10 mg/kg, respectively) and blood was drawn by cardiac puncture. Whole blood was clotted for 30–60 minutes then centrifuged at 2000 × g for 20 min. TIF and sera supernatants were analyzed using a MILLIPLEX MAP Mouse Cytokine/Chemokine Panel as per manufacture’s instructions (EMD Millipore).
3
Quantification of Cytokine Production
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Cytokine production was quantified by Milliplex MAP Mouse Cytokine/Chemokine Panel (Merck Millipore), following the manufacturer's instruction. The results were expressed as pg·mg protein−1.
4
Proteomic Analysis of Colonic Inflammation
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Colonic samples (n = 5–6/group per time point) were collected from C57BL/6 female mice receiving 2% DSS or 2% DSS + TUS. Collection was performed at days 0, 3, 5, 7, 9, 11 and 14. After PBS 1× cleaning, samples were snap frozen and processed for further proteomic analysis. Briefly, frozen colonic samples were homogenized in cell lysis buffer (1 mM EDTA, 150 mM NaCl, 0.05% Tween-20 and 20 mM Tris-HCl in ultrapure water) containing Pierce Protease Inhibitor Tablets (Thermo Scientific, Waltham, MA) and 1.0 mm Zirconium Beads. Homogenates were centrifuged at 14,000 rpm at 4 °C for 20 min and the supernatant was collected. The process was repeated two times and aliquots were stored at −80 °C. Bicinchoninic acid assay (BCA – Thermo Scientific, Waltham, MA) was used for protein quantification and samples were further diluted to 1 mg/mL of total protein. MILLIPLEX Map Mouse Cytokine/Chemokine Panel (EMD Millipore, Billerica, MA) was used for proteomic analysis of colonic homogenates according to manufacturer specifications in a Bio-Plex 200 (Bio-Rad Laboratories, Hercules, CA). The same control samples (day 0) were used for multiplex ELISA experiments in the 2% DSS and 2% DSS + TUS exposed mice. Further analysis of colonic TGFβ (Thermo Scientific, Waltham, MA) and HSP70 (R&D Systems, Minneapolis, MN) were performed using ELISA Streptavidin-HRP assay.
5
Colon Cytokine Profiling in DSS-Induced Colitis
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Colon samples from animals (n = 6/each time point) receiving 3% DSS were snap frozen and later homogenized for protein extraction. Briefly, frozen colon samples were processed in cell lysis buffer containing 150 mmol/L NaCl, 1 mmol/L EDTA, 20 mmol/L Tris-HCl and 0.05% Tween-20, with addition of protease inhibitor (Thermo Scientific, Waltham, MA, United States) and 1.0 mm Zyrconium Beads. Samples were centrifuged twice at 14000 r/min at 4 °C for 20 min and supernatant was collected. Aliquots were kept at -80 °C until further analysis. Samples were quantified through bicinchoninic acid assay (BCA - Thermo Scientific, Waltham, MA, United States) and diluted to a final concentration of 1 mg/mL of total protein. Colon homogenates were analyzed by MILLIPLEX Map Mouse Cytokine/Chemokine Panel (EMD Millipore, Billerica, MA, United States) using Bio-Plex 200 (Bio-Rad) according to manufacturer specifications.
6
Multiplex Cytokine Profiling in Plasma
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By using a customized detection MILLIPLEX MAP Mouse Cytokine/Chemokine panel (Merck Millipore, Billerica, MA), the following 13 cytokines/chemokines were evaluated in plasma-EDTA samples: eotaxin, G-CSF (granulocyte-colony stimulating factor), IL (interleukin)-4, IL-5, IL-6, IL-10, IL-12(p70), IL-15, IL-17, IP-10 (interferon gamma-induced protein 10), KC/CXCL1 (keratinocyte-derived cytokine), LIX/CXCL5 (lipopolysaccharide-inducible CXC chemokine), and MIG/CXCL9 (monokine induced by gamma interferon). These analyses were outsourced to certified laboratories (Department of Experimental Evolutionary Biology, University of Bologna and Labospace Srl).
7
Multiplex Cytokine Quantification in Mice
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The concentration of IFN-γ, IL-6, and IL-10 cytokines was determined in the plasma of experimental mice by the MILLIPLEX MAP Mouse Cytokine/Chemokine Panel (Merck Millipore, MA, United States) according to the manufacturer’s instructions and analyzed using the Bio-Plex System (Bio Rad Laboratories, CA, United States).
8
Multiplex Cytokine Quantification
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Plasma concentrations of IL-6, TNFα, MCP-1, IL-1β, IFN-γ, IL-10 were quantified using a Milliplex MAP Mouse Cytokine/Chemokine Panel (# MCYTOMAG-70K, Millipore, Billerica, MA). The assays were performed according to the manufacturer’s instructions. Standards and samples were analyzed on a LuminexR® apparatus (Bio-Plex 200, BioRad, München, Germany) using the BioPlex Manager Software (Version 5, BioRad, Hercules, CA).
9
Cytokine and Chemokine Profiling in Ear Inflammation
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Ears were removed 24 h after challenge and each ear was weighted and placed in 0.5 ml buffer (0.9% Saline with 0.01% Triton X-100 (Sigma) + 1 protease inhibitor cocktail tablet (Complete EDTA-free from Roche)) on ice. The biopsies were subsequently homogenized and centrifuged 15 min for 10,000g at 4°C. The supernatants were centrifuged once more before being frozen at −80° until use. Supernatants were analyzed with MILLIPLEX MAP Mouse Cytokine/Chemokine Panel (Millipore, Billerica, MA) by the Luminex detection method. Supernatants were analyzed for the following cytokines and chemokines: MIP-2 (CXCL2), MCP-1 (CCL2), KC (CXCL1), MIG (CXCL9), RANTES (CCL5), LIX (CXCL5), IL-4, IL-1β, IFNγ, IL-6, IL-10, G-CSF, and TNFα.
10
Quantifying Inflammatory Cytokines in Mouse Plasma
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The mouse plasma was centrifuged and the supernatant was used to determine the level of inflammatory cytokines. The concentrations of TNF-α, IL-6, and IL1-β were measured by a Milliplex MAP Mouse Cytokine/Chemokine Panel (Millipore Corporation, MA, USA) on the Luminex-200 platform. Data analysis was performed with Milliplex Analyst 5.1 software (Millipore Corporation, MA, USA) [20 (link)].
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