The Human IL8 kit is designed for the quantification of human IL8 release in cell supernatant.
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  • No-wash
  • Validated on PBMC
  • Under 2h bench time
The Human IL8 kit is designed for the quantification of human IL8 release in cell supernatant.
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In stock

Overview

Also called CXCL8, IL8 is a chemokine mainly produced by macrophages, T cells, and neutrophils. IL8 acts as a chemoattractant for neutrophils, and promotes infiltration and activation at inflammation sites. IL8 is involved in several cancer types due to its ability to promote angiogenesis and cell proliferation, as well as to inhibit apoptosis.

Assay principle

Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection by HTRF® reagents (384-well low-volume white plate or Cisbio low-volume 96-well plate in 20 µl). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run up to a 1536-well format by simply resizing each addition volume proportionally.

Assay data analysis

The 4 Parameter Logistic (4PL) curve is commonly recommended for fitting an ELISA standard curve. This regression enables the accurate measurement of an unknown sample across a wider range of concentrations than linear analysis, making it ideally suited to the analysis of biological systems like cytokine releases. To fully understand how to deal with HTRF data processing and also 4PL 1/y² fitting, please visit this page.

Cisbio also worked with Myassays.com to help you in your data analysis. By clicking on this link, you’ll be able to access a free online software to run your IL8 analysis.

Technical specifications of human IL8 kit

Sample size 16 µL
Final assay volume 20 µL
Kit components Lyophilized standard, frozen detection antibodies, buffers &protocol.
LOD &LOQ (in Diluent) 6 pg/mL &32 pg/mL
Range 32 – 4,000 pg/mL
Time to result 1h at RT
Calibration NIBSC (89/520) value (IU/mL) = 0,01 x HTRF hIL8 value (pg/mL)
Species Human only

Intra and inter assay

Intra-assay (n=24)

Sample Mean [IL8] (pg/mL) CV
1 58 9%
2 314 4%
3 2074 2%
Mean CV 5%

Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.

Inter-assay (n=4)

Sample [IL8] (pg/mL) Mean (delta R) CV
1 78 273 2%
2 376 1504 12%
3 1818 5990 5%
Mean CV 6.3%

Each of the samples was measured in 4 different experiments, and % CV was calculated for each sample.

Dilutional linearity

Sample
Dilution factor[IL8] expected (pg/mL)[IL8] detected (pg/mL)Recovery
11-2015-
4.3469489104%
7.4271292108%
10.7188210111%
Mean108%
21-399-
4.3938591%
7.45456103%
Mean97%

The excellent % of recovery obtained from these experiments show the good linearity of the assay.

Spike and recovery

Sample
[IL8] added (pg/mL)[IL8] expected (pg/mL)[IL8] detected (pg/mL)Recovery
15,0005,1795,00897%
25,0006,0475,68294%

​The same amount of recombinant cytokine was added to 2 different serum samples, and the set of responses obtained from a standard curve was compared to the calculated expected values. The ~ 100% of recovery found validates the sample matrix used for this assay.

Inhibition of IL8 secretion in THP1 cells with JTE607

THP1 cells plated at 100 kcells/well were incubated with increasing concentrations of JTE 607 for 18h, then stimulated for 3 h with 2 µg/mL LPS. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human IL8 Assay.

IL8 secretion in PBMCs stimulated with LPS

PBMC plated at 50, 100, 200, and 400 kcells/well were stimulated for 3 h with increasing concentrations of LPS (0, 0.02, 0.2, 2 µg/mL). 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human IL8 Assay.

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Validated protocol - Technical Notes

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