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.

HTRF human IL-2 standard curve 

The IL-2 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 the % CV was calculated for each sample.

Inter-assay (n=4)

Each of the samples was measured in 4 different experiments, and the % CV was calculated for each sample.

Effect of dexamethasone on PBMC induced IL-2 release

PBMC plated at 25, 50 and 100 kcells/well were stimulated for 18h with 50 ng/mL of PMA/ 1 µg/mL ionomycine and a co-treatment with dexamethasone (50 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 IL-2 Assay kit.

Anti-CD3 and CD28 Ab dose response effect on IL-2 secretion

Jurkat cells plated at 200 kcells/well were stimulated for 24 h with increasing concentrations of anti CD3 and anti-CD28 antibodies : 0, 0.5, 1, 10 µg/mL for the anti CD3, and 0, 1, 5, 20 µg/mL for the anti CD28. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP human IL-2 assay.

IL-2 secretion in PBMCs cells from different donors

PBMC plated at 100 kcells/well were stimulated for 18h with 50 ng/mL of PMA/ 1 µg/mL ionomycine. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human IL-2 Assay kit.