Human CCL5 (RANTES) Assay Kit

CCL5, also known as RANTES (regulated upon activation, normal T-cell expressed and secreted), is a chemokine mainly secreted by T lymphocytes, macrophages, platelets, synovial fibroblasts, tubular epithelium and certain types of tumor cells. CCL5 plays an important role on inflammatory sites by recruiting T cells, macrophages, eosinophils, and basophils. When secreted with IL-2 and IFN-g, CCL5 also induces the activation and proliferation of natural killer cells.

The functions of CCL5 in a tumor environment is still unclear, as CCL5 can act to induce an immune response against the tumor but is also associated with cancer progression and metastasis. In infectious diseases, CCL5 has been described as inhibiting HIV entry into target cells.

Cisbio's Human CCL5 kit is a homogeneous, add and read assay to measure CCL5 secretion in cell supernatant. It offers a fast and 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) 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
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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Cytokines assay principle add

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Cytokines assay principle read

(1) Assay data analysis:

  • "Guidelines for data reduction" button will show you how to deal with HTRF data processing and 4PL 1/y2 fitting.
  • "MyAssays" button will direct you to a free online software able to run CCL2 analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

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%

Assay validation

1. CCL5 secretion in PBMCs stimulated with LPS, IL1β and TNFα.

PBMCs were plated at 100,000 cells/well in a 96 well plate, and stimulated for 24h with LPS, IL1β and TNFα 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 the Human CCL5 Assay Kit.

2. CCL5 secretion in LX2 stimulated with TGFβ.

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 TGFβ at 0.5ng/ml. Next, 16µl of supernatant were transferred into a 384 well (SV) white detection plate to be analyzed by the Human CCL5 Assay Kit.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human CCL5 kit - 500 tests62HCCL5PEG
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Human CCL5 kit - 10,000 tests62HCCL5PEH
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Companion products

DescriptionCat. noProduct insertMSDS
Human CCL5 kit - 3 Standards62HCCL5CD3
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Human CCL5 kit - Standard62HCCL5CDA
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