HTRF Human TNF-α High Performance Detection Kit, 10,000 Assay Points

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.

The HP human TNF alpha standard curve is generated in the assay Diluent 1, provided in the kit.

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

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample.

Cross reactivities were assessed using recombinant proteins from the TNF alpha cytokine family. Proteins were tested up to 5,500 pg/mL, and standard curves were generated for each. Signals were interpolated on the assay standard curve to check concentrations.

PBMCs plated at 50, 100, 200, and 400 kcells/well were stimulated for 3h with LPS at 0.2 µg/mL. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human TNF alpha assay Kit.

THP-1 cells plated at 100 kcells/well were stimulated for 18h with increasing concentrations of the inhibitor JTE 607 in presence of 2 µg/mL LPS. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using HTRF HP Human TNF alpha assay kit.

PBMCs plated at 200 and 400 kcells/well were stimulated for 18h with increasing concentrations of the inhibitor JTE-607 in presence of 2 µg/mL LPS. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human TNF alpha assay kit.