Human IL8 Assay Kit

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 inhibit apoptosis.
Cisbio's Human IL8 kit is a homogeneous, add and read assay to measure IL8 secretion in supernatant. It offers a fast, efficient alternative to ELISA.

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
Download Insider tips for PBMC isolation Technical Note
Download cytokine release from fresh blood samples application notes

Assay principle

The assay protocol, using a Cisbio 96-well low volume or a 384-well small volume white plate (20 µL final), is described below. Cell supernatant, sample or standard are dispensed directly into the assay plate for the detection by HTRF® reagents. 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 in 96 to 1536-well plates by simply resizing each addition volume proportionally.

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.

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(1) Assay data analysis:

  • "Guidelines for data reduction" button will show you how dealing with HTRF data processing and 4PL 1/y2 fitting.
  • "MyAssays" button will direct you to a free online software able to run IL8 analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

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.

Accuracy

Dilutional linearity

Sample Dilution factor [IL8] expected (pg/mL) [IL8] detected (pg/mL) Recovery
1 1 - 2015 -
4.3 469 489 104%
7.4 271 292 108%
10.7 188 210 111%
Mean 108%
2 1 - 399 -
4.3 93 85 91%
7.4 54 56 103%
Mean 97%

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
1 5,000 5,179 5,008 97%
2 5,000 6,047 5,682 94%

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.

Assay validation

1. IL8 secretion in THP1 cells stimulated with LPS: kinetic of stimulation

THP1 cells plated @ 50 kcells/well were stimulated for 3h & 18h with LPS used at 2 concentrations: 0.02 & 0.2 µg/mL.
16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IL8 Assay Kit.

2. Inhibition of IL8 secretion in THP1 cells with JTE607

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

3. IL8 secretion in PBMCs stimulated with LPS: cell densities

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

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human IL8 kit - 500 tests62HIL08PEG
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Human IL8 kit - 10,000 tests62HIL08PEH
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Human IL8 kit - 1 x 96 tests62HIL08PET
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Companion products

DescriptionCat. noProduct insertMSDS
Human IL8 kit - 3 Standards62HIL08CD3
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Human IL8 kit - Standard62HIL08CDA
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