Human Chemokine (C-C motif) Ligand 3, CCL3 ELISA Kit (CKERS-CCL3-022H)

Regulatory status: For research use only, not for use in diagnostic procedures.

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Size
15plates, 45plates
Species Reactivity
Human
Intended Use
Human MIP-1α ELISA development kit contains the key components required for the quantitative measurement of natural and/or recombinant MIP-1α in a sandwich ELISA format within the range of 16-3,000 pg/mL. Using the ELISA protocol described below, this kit provides sufficient reagents to assay MIP-1α in approximately 1,500 ELISA plate wells.
Contents of Kit
1. PBS: Dilute 10xPBS to 1xPBS, pH 7.2 in sterile water.
2. Wash Buffer: 0.05% Tween-20 in PBS
3. Block Buffer: 1.0% BSA in PBS
4. Diluent: 1.0% BSA in PBS
5. ELISA microplates (Thermo Fisher Cat. # 456529);
6. BSA (Sigma Cat. # A-7030);
7. Stop Solution 2 M Sulfuric Acid (Sigma Cat. # 339741);
8. Dulbecco's PBS [10x] (Gibco BRL Cat. # 14200-075).
Storage
Unopened Kit Store at 2-8°C. Do not use past kit expiration date. For more detailed information, please download the document on our website.
General Description
This locus represents a small inducible cytokine. The encoded protein, also known as macrophage inflammatory protein 1 alpha, plays a role in inflammatory responses through binding to the receptors CCR1, CCR4 and CCR5. Polymorphisms at this locus may be associated with both resistance and susceptibility to infection by human immunodeficiency virus type 1.
Standard Curve
Reconstitution And Storage
Capture Antibody: 72 μg of antigen-affinity purified goat anti-MIP-1α. Centrifuge vial prior to opening. Reconstitute in 1.0 mL PBS for a concentration of 72 μg/mL. Following reconstitution the Capture antibodies may be stored at 2-8°C for up to 6 months. For long term storage it is recommended to aliquot into working volumes and store at -70°C in a manual defrost freezer.
Detection Antibody: 36 μg of biotinylated antigen-affinity purified goat anti-MIP-1α. Centrifuge vial prior to opening. Reconstitute in 1.0 mL sterile 1.0% BSA in PBS for a concentration of 36 μg/mL. Following reconstitution the Detection antibodies may be stored at 2-8°C for up to 6 months. For long term storage it is recommended to aliquot into working volumes and store at -70°C in a manual defrost freezer.
Human MIP-1α Standard: 45 ng of recombinant MIP-1α. Centrifuge vial prior to opening. Reconstitute in 0.5 mL sterile 1.0% BSA in PBS for a concentration of 90 ng/mL. The Standard may be stored at 2-8°C for one month or aliquoted and stored at -70°C for up to three months in a manual defrost freezer.
UltraAvidin-HRP Conjugate: 40 μL vial. Upon receipt, UltraAvidin-HRP conjugate should be stored at 2-8°C, DO NOT FREEZE.
TMB Liquid Substrate: Aspirate and wash plate 4 times. Add 100 μL of TMB HRPO Microwell Substrate Standard Kinetic One Component "Ready Use" to each well. Incubate at room temperature and monitorcolor development with an ELISA plate reader at 450 nm with wavelength correction set at 540 nm or 570 nm. The reaction may be stopped after 15-20 minutes by adding 100 μL of 2 M sulfuric acid to each well.

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References


Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9

FRONTIERS IN IMMUNOLOGY

Authors: Schenten, Veronique; Plancon, Sebastien; Jung, Nicolas; Hann, Justine; Bueb, Jean-Luc; Brechard, Sabrina; Tschirhart, Eric J.; Tolle, Fabrice

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca2+-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1 alpha, IL-1 beta, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling.

Circulating inflammatory markers and colorectal cancer risk: A prospective case-cohort study in Japan

INTERNATIONAL JOURNAL OF CANCER

Authors: Song, Minkyo; Sasazuki, Shizuka; Camargo, M. Constanza; Shimazu, Taichi; Charvat, Hadrien; Yamaji, Taiki; Sawada, Norie; Kemp, Troy J.; Pfeiffer, Ruth M.; Hildesheim, Allan; Pinto, Ligia A.; Rabkin, Charles S.; Tsugane, Shoichiro

Blood levels of inflammation-related markers may reveal molecular pathways contributing to carcinogenesis. To date, prospective associations with colorectal cancer (CRC) risk have been based on few studies with limited sets of analytes. We conducted a case-cohort study within the Japan Public Health Center-based Prospective Study Cohort II, comparing 457 incident CRC cases during median 18 years follow-up with a random subcohort of 774 individuals. Baseline plasma levels of 62 cytokines, soluble receptors, acute-phase proteins, and growth factor markers were measured using Luminex bead-based assays. We estimated hazard ratios (HRs) associating each marker with CRC risk by Cox proportional hazards models adjusted for potential confounders. Subanalyses compared cases by years after blood draw (vs. >= 5) and anatomical subsite (colon vs. rectum). Linear trends in quantiles of four C-C motif ligand (CCL) chemokines, one C-X-C motif ligand (CXCL) chemokine, and a soluble receptor were nominally associated with CRC risk based on p(trend) < 0.05, but none met false discovery rate corrected statistical significance. HRs for the 4th vs. 1st quartile were: 1.69 for CCL2/MCP1, 1.61 for soluble tumor necrosis factor receptor 2, 1.39 for CCL15/MIP1D, 1.35 for CCL27/CTACK, 0.70 for CXCL6/GCP2 and 0.61 for CCL3/MIP1A. Among cases diagnosed 5+ years after enrollment, CCL2/MCP1, CCL3/MIP1A and CXCL6/GCP2 retained nominal statistical significance. There were no significant differences in associations between colon and rectum. Our findings implicate chemokine alterations in colorectal carcinogenesis, but require replication for confirmation. Noninvasive chemokine assays may have potential application in colorectal cancer screening and etiologic research.

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