The Human CCL5 (RANTES) kit is designed for the quantification of human CCL5 release in cell supernatant.
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  • No-wash
  • Low sample consumption
  • Under 2h bench time
The Human CCL5 (RANTES) kit is designed for the quantification of human CCL5 release in cell supernatant.
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In stock

Overview

CCL5, also known as RANTES (regulated upon activation, normal T-cell expressed and secreted), is a chemokine mainly secreted by T lymphocytes, macrophages, platelets, and certain types of tumor cells. RANTES plays an important role in inflammatory sites by recruiting T cells and macrophages. When secreted with IL-2 and IFN-g, RANTES also induces the activation and proliferation of natural killer (NK) cells. The function of RANTES in oncology is still unclear, but in infectious diseases RANTES has been described as inhibiting HIV entry into target cells.

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 CCL5 analysis.

Technical specifications of human CCL5 kit

Sample size 16 µL
Final assay volume 20 µL
Kit components Lyophilized standard, frozen detection antibodies, buffers &protocol
LOD &LOQ (in Diluent) 7 pg/mL &18 pg/mL
Range 18 – 2,000 pg/mL
Time to result ON @ RT
Calibration NIBSC (92/520) value (IU/mL) = 0,015 x HTRF hCCL5 value (pg/mL)
Species Human only

Intra and Inter assay

Intra assay (n=24)

Sample Mean [CCL5] (pg/mL) CV
1 74 7%
2 417 7%
3 1047 6%
Mean CV 7%

Inter assay (n=4)

Sample [CCL5] (pg/mL) Mean (delta ratio) CV
1 62 320 12%
2 256 1214 13%
3 1008 4405 5%
Mean CV 10%

CCL5 secretion in PBMCs stimulated with LPS, IL1ß and TNFa

PBMCs were plated at 100,000 cells/well in a 96 well plate, and stimulated for 24h with LPS, IL1ß and TNFa respectively used at 200 µg/mL, 10 ng/mL and 10 ng/mL. Supernatants were diluted to 1/3, and 16 µL of the diluted samples were transferred into a 384 well (SV) white detection plate to be analyzed by with the Human CCL5 Assay.

CCL5 secretion in LX2 stimulated with TGFß

LX2 cells were plated at 400,000 cells/well in a 6 well plate, cultured for 3 days, then starved for 1 day before 48 hours of stimulation with TGFß at 0.5ng/ml. Next, 16 µL of supernatant were transferred into a 384 well (SV) white detection plate to be analyzed with the Human CCL5 Assay.

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