HTRF Human and Mouse TGF-β1 Detection Kit, 10,000 Assay Points

Activation step

The samples and cell culture supernatants contain TGF beta 1 and a TGFß1-LAP complex, which require an acid activation step followed by a neutralization in order to be detected (sample activation can follow one of the protocols described on the right). The reagents needed for this activation step are provided in the kit for maximum convenience. Standards and activated samples are dispensed directly into the detection (white, low-volume) plate for the detection by HTRF® reagents.

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 Revvity 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.

Intra assay (n=24)

Inter assay (n=4)

Human TGFß1 secretion in PBMC cells stimulated with Ionomycin & PMA

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.

Mouse TGFß1 secretion from immortalized kupffer cells

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.