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hIL-8 QKit (1 Kit)
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hIL-8 QKit (1 Kit)

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品牌: R&D Systems
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Product Summary

The Quantikine Human IL-8 Immunoassay is a 3.5 hour solid phase ELISA designed to measure human IL-8 in cell culture supernates, serum, and plasma. It is based on antibodies raised against the 72 aa variant of human IL-8 derived from E. coli. It is calibrated with the same recombinant factor. This immunoassay accurately quantitates recombinant human IL-8. Measurement of natural human IL-8 or the 77 aa variant of human IL-8 gave results parallel to the standard curves obtained using the E. coli-expressed Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human IL-8.

Sample Values

Serum/Plasma - Thirty-four samples from apparently healthy volunteers were evaluated for the presence of human IL-8 in this assay. All samples measured less than the lowest human IL-8 standard, 31.3 pg/mL. No medical histories were available for the donors used in this study.

Cell Culture Supernates - Human peripheral blood mononuclear cells (1 x 106 cells/mL) were cultured in RPMI supplemented with 10% fetal bovine serum, 50 μM b-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate and stimulated with 10 μg/mL PHA. Aliquots of the culture supernate were removed on days 1 and 5 and assayed for levels of human IL-8.

ConditionDay 1 (pg/mL)Day 5 (pg/mL)
Unstimulated 27,00033.000
Stimulated 73,000102,000

Recovery

The recovery of IL-8 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample TypeAverage % RecoveryRange %
Cell Culture Media (n=5)9885-114
Citrate Plasma (n=5)10595-114
EDTA Plasma (n=5)10397-111
Heparin Plasma (n=5)10292-107
Serum (n=5)9888-106

Linearity

To assess the linearity of the assay, samples were spiked with high concentrations of IL-8 in various matrices and diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Human CXCL8 ELISA/IL-8 ELISA Linearity

Scientific Data

Human CXCL8 ELISA/IL-8 ELISA Cell Culture Supernate Standard Curve

Human CXCL8 ELISA/IL-8 ELISA Serum/Plasma Standard Curve

Human IL-8/CXCL8 Quantikine ELISA Kit RT-qPCR for a set of cytokines (CSF2, IL6, IL8 and IL1B) and chemokines (CXCL1 and CXCL6) of total RNA samples derived from mono- and co-cultures in a transwell assay.The respective co- (+) or mono- (-) culture is indicated on the X-axis. The respective analyzed cytokine or chemokine is indicated in the header of each graph. Expression values are shown in arbitrary units (AU) and have been normalized to beta-2 microglobulin (B2M) mRNA copies. Experiments were performed in triplicates (n = 3). Statistical analysis was performed on the mean values by unpaired comparison of mono-cultured HDF and co-cultured HDF RNA samples by using Student’s t-test (**p<0.01, ***p<0.001; n.s.: not significant). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25919140), licensed under a CC-BY license. Not internally tested by R&D Systems.

Assay Procedure

Refer to the product for complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
  1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
  2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

  3. 100 µL Assay Diluent
  4.   Add 100 µL of Assay Diluent to each well.

  5. 50 µL Standard, Control, or Sample
  6.   Add 50 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
  7.   Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.

  8. 100 µL Conjugate
  9.   Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour.
  10.   Aspirate and wash 4 times.

  11. 200 µL Substrate Solution
  12.   Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.

  13. 50 µL Stop Solution
  14.   Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.

Human IL-8/CXCL8 Quantikine ELISA Kit Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
3.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (50 uL), Serum (50 uL), EDTA Plasma (50 uL), Heparin Plasma (50 uL), Citrate Plasma (50 uL)
Sensitivity
7.5 pg/mL
Assay Range
31.2 - 2,000 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Citrate Plasma)
Specificity
Natural and recombinant human IL-8
Cross-reactivity
< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
Interference
No significant interference observed with available related molecules.
背景
别名
3-10C,AMCF-I,C-X-C motif chemokine 8,CXCL8,CXCL8SCYB8,Emoctakin,GCP1,GCP-1TSG-1,IL8,IL-8,interleukin 8,K60,LAI,LECT,LUCT,LYNAP,MDNCF,MDNCFb-ENAP,member 8,MONAP,MONAPGCP1,NAF,NAP1,NAP-1NAP1,NCF,Neutrophil-activating protein 1,Protein 3-10C,T cell chemotactic factor,T-cell chemotactic factor,TCF,TSG1,Interleukin 8
背景

Background: IL-8/CXCL8

Interleukin-8 (IL-8), also known as IL-8, GCP-1, and NAP-1, is a heparin-binding 8-9 kDa member of the alpha, or CXC family of chemokines. There are at least 15 human CXC family members that all adopt a three beta -sheet/one alpha -helix structure. Most CXC chemokines show an N-terminal Glu-Leu-Arg (ELR) tripeptide motif. IL-8 circulates as a monomer, homodimer, and heterodimer with CXCL4/PF4. The monomer is considered the most bio-active, while the heterodimer can potentiate PF4 activity. IL-8 oligomerization is modulated by its interactions with matrix and cell surface glycosaminoglycans (GAGs). Mature human IL-8 shares 65-69% amino acid (aa) identity with canine, feline, and porcine IL-8. There is no IL-8 gene counterpart in rodent. 


Multiple isoforms of IL-8 are generated through both alternative splicing and differential proteolytic cleavage. In humans, alternative splicing generates an iso-form with an eleven aa substitution at the C-terminus. Proteolytic processing results in N-terminal truncation of IL-8 and is likely a cell-specific event. For example, fibroblasts and endothelial cells generate the 1-77 form by cleaving IL-8 following Glu21, while monocytes and lymphocytes generate the 6-77 form by cleaving following Leu25. These truncated forms generally show increased bioactivity, particularly through the CXCR1 receptor. IL-8 can also undergo citrullination on Arg27 of the precursor, a modification that increases its half-life and ability to induce leukocytosis. A wide variety of cells secrete IL-8 including monocytes and neutrophils, fibroblasts and keratinocytes, mast cells, visceral smooth muscle cells, dendritic cells, type II great alveolar cells, and endothelial cells. 

IL-8 bioactivity is mediated through two G-protein-coupled receptors, termed CXCR1/IL-8 RA and CXCR2/IL-8 RB. CXCR1 is 45-50 kDa in size and is used almost exclusively by IL-8. CXCR2 is 35-40 kDa in size and is used by nearly all CXC chemokines. Both CXCR1 and CXCR2 constitutively associate into functional homodimers. They can also heterodimerize, but these complexes dissociate following IL-8 binding. CXCR2 responds to low concentrations of IL-8 and is principally associated with chemotaxis and MMP-9 release. CXCR1, in contrast, responds to high concentrations of IL-8 and is associated with respiratory burst and phospholipase D2 activation. Thus, CXCR2 ligation induces leukocyte adhesion to activated vascular endothelium and migration to sites of inflammation, while CXCR1 ligation primes neutrophil antimicrobial activity. IL-8 can also form a complex with Serpin A1/alpha-1 Antitrypsin, and this prevents IL-8 interaction with CXCR1. 

In addition to its pro-inflammatory effects, IL-8 is involved in angiogenesis and the pathogenesis of atherosclerosis and cancer. It induces VEGF expression in vascular endothelial cells and functions as an autocrine factor for EC growth and angiogenesis. It is upregulated in atherosclerotic lesions and is elevated in the serum and cerebrospinal fluid following myocardial infarction. In cancer, IL-8 promotes epithelial-mesenchymal transition as well as tumor cell invasiveness and metastasis.
Long Name:
Interleukin 8
Entrez Gene IDs:
3576 (Human); 396880 (Porcine); 403850 (Canine); 493836 (Feline)
Alternate Names:
3-10C; AMCF-I; C-X-C motif chemokine 8; CXCL8; CXCL8SCYB8; Emoctakin; GCP1; GCP-1TSG-1; IL8; IL-8; interleukin 8; K60; LAI; LECT; LUCT; LYNAP; MDNCF; MDNCFb-ENAP; member 8; MONAP; MONAPGCP1; NAF; NAP1; NAP-1NAP1; NCF; Neutrophil-activating protein 1; Protein 3-10C; T cell chemotactic factor; T-cell chemotactic factor; TCF; TSG1
制备和贮存
保存方式

Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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货号:
D8000C
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