Human IL8 kit HTRF®

Name: Human IL8 kit
Brand: Cisbio
SKU: 40421
Availability: InStock

Associated products

The HTRF human IL8 kit is designed for the quantification of human IL8 release in cell supernatant.

No-wash
Validated on PBMC
Under 2h bench time

Download the protocols

The HTRF human IL8 kit is designed for the quantification of human IL8 release in cell supernatant.

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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.

Assessment of serum samples often requires enhanced sensitivity. In some cases, AlphaLISA assays may have sufficient sensitivity to enable the detection of low levels of analytes in serum or plasma. Check out the Human IL-8 AlphaLISA kit’s analytical performances for more information or learn more about AlphaLISA. When assaying, always follow the recommended protocol and avoid highly haemolyzed samples.

Benefits

VALIDATED ON PBMC
TLR PATHWAY READOUT

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
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.

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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