No-wash kit to quantify released Human CCL2 (MCP1)
Human and mouse TGF beta 1 kit
- Validated on PBMC
- Under 2h bench time
TGF beta 1 (Transforming Growth Factor Beta-1) is considered one of the major cytokines involved in the regulation of extracellular matrix (ECM) synthesis and degradation, as well as a major profibrotic factor. TGF beta 1 pre-proprotein is processed to generate a latency-associated peptide (LAP) and a mature TGFß1 peptide, which remain associated through strong non-covalent interactions. An activation step (MMP2 cleavage or acid activation) is required to release the active form of TGF beta 1 from the TGFß1-LAP complex.
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 TGF beta 1 analysis.
|Sample size||16 µL|
|Final assay volume||20 µL|
|Kit components||Lyophilized standard, frozen detection antibodies, buffers &protocol|
|LOD &LOQ (in Diluent)||4 pg/mL &19 pg/mL|
|Range||19 2,000 pg/mL|
|Time to result||ON at RT|
|Calibration||NIBSC (89/514) value (U/mL) = 0.017 x HTRF hTGFß value (pg/mL)|
|Species||Human, mouse, bovine (use of FCS is possible up to 5% max), others expected (based on sequences similarities). No detection of chicken TGFß1.|
|Specificity||There is no cross-reactivity with human TGFß2 and TGFß3.|
Intra assay (n=24)
|Sample||Mean [TGFβ1] (pg/mL)||CV|
Inter assay (n=4)
|Sample||[TGFβ1] (pg/mL)||Mean (delta ratio)||CV|
Human PBMCs plated at 125 kcells/well (96 wells plate) in RPMI containing 4% FCS were stimulated for 18 h with Ionomycin (1 µg/mL) and PMA ranging from 0.5 to 50 ng/mL. After transfer into polypropylene microtubes, 50 µL of supernatants were treated with the acid activation reagent (5 µL, 10 min, room temperature) then with the neutralization reagent (5 µL). 16 µL were transferred into a white detection plate. Recommended controls include complemented media alone and unstimulated cells in complemented medium to determine TGF beta in stimulation media and baseline concentrations respectively.
Immortalized mouse Kupffer cells (ImKC) were plated at 650 kcells/well (24 wells plate) in RPMI containing 4% FCS. After 24 h resting, the cells were stimulated for 16 h with increasing concentrations of LPS ranging from 0.05 to 5 µg/mL (final volume 500 µL/well). 100 µL of supernatants were acid activated (10 µL) for 10 min at room temperature then neutralized (10 µL). 16 µL of activated supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human TGF beta 1 Assay.
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