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 in up to a 1536-well format by simply resizing each addition volume proportionally.

Assay principle - p40 family

The assay is compatible with human samples only, and is highly specific for p40, cross-reacting with all p40-containing cytokines such as IL12, IL23, p40 monomer, and p40-p40 (p80) dimer.

Standard curve

The IL12/23 p40 standard curve is generated in the assay Diluent 5 provided in the kit.

Intra-assay (n=24)

Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.

Samples were cell culture supernatants from stimulated THP1 cells.

Inter-assay (n=3 experiments)

Each of the samples was measured in 3 independent experiments (3 days), and % CV was calculated for each sample. Samples were cell culture supernatants from stimulated THP1 cells.

IL12/23 p40 secretion from LPS-stimulated THP1 human monocytes

The human monocyte cell line THP1 was used for an IL12/23-p40 secretion study using LPS as an agonist. THP1 cells were cultured in 10% FCS supplemented RPMI1640 medium at 37°C, 5% CO2. 50 µL of THP1 cells in suspension were transferred into a 96-well cell-culture plate at densities of 200 and 400 k cells/well. Cells were stimulated for 24h with increasing doses of LPS in a final volume of 100 µL.  16 µL of supernatants were then transferred into a white detection plate (384 low volume), and 4 µL of the HTRF Human IL12/23-p40 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. As expected, increasing doses of LPS led to incremental releases of IL12/23-p40 cytokines. The concentrations measured were lower using the highest density, indicating that the 200k density was the best compromise to study the secretion of the cytokines using this cell model in suspension.