HTRF Human Pro IL-1β Detection Kit, 10,000 Assay Points

Protocol of the HTRF human pro IL-1β assay

The HTRF pro IL-1β assay can be run in a 96- or 384-well low volume white detection plate (20 µL final). As described here, samples (cell lysates) or standards are dispensed directly into the assay plate for the detection of pro IL-1β by HTRF reagents. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Intra-assay precision table

All of the 3 samples wwere measured 24 times, and the % CV was calculated for each. The samples were cell lysates from LPS stimulated THP-1.

Inter-assay precision table

All of the samples wwere measured in 3 independent experiments (3 days), and the % CV was calculated for each. The samples were cell lysates from LPS stimulated THP-1.

Pro IL-1β expression in LPS stimulated THP1

THP1 cells were plated at 25, 50,100, and 200 kcells/well and were stimulated for 3h with LPS at 1 µg/mL. After treatment, the medium was removed and cells were lysed with 40µL lysis buffer #1 for 30 minutes under gentle shaking. Then 16 µL of cell lysates were transferred into a white detection plate (384 low volume) to be analyzed with the human pro IL-1β assay.

Pro IL-1β modulation using nigericin on LPS stimulated THP1 cells

THP1 cells were seeded in a 96-well culture plate (100,000 cells) and were treated with 1µg/mL LPS for 3 hours. Cells were then treated with increasing concentrations of Nigericin for 2 hours. After treatment, the medium was collected, and cells were lysed with 40µL lysis buffer #1 for 30 minutes under gentle shaking. Next, the cell lysates were tested using the HTRF pro IL-1β kit and the medium was tested using the HTRF IL-1β high performance detection kit (part 62HIL1B2PEG).

As expected, Nigerinicin induced the cleavage of pro IL-1β in LPS stimulated THP1 cells and the secretion of IL-1β in cell medium.

Pro IL-1β modulation kinetics using nigericin

THP1 cells were seeded in a 96-well culture plate (100,000 cells) and were treated with 1µg/mL LPS for 3 hours. Cells were then treated with 12.5µM of Nigericin for 15 to 120 minutes. After treatment, the medium was collected, and cells were lysed with 40µL lysis buffer #1 for 30 minutes under gentle shaking. Next, the cell lysates were tested using the HTRF pro IL-1β kit and the medium was tested using the HTRF IL-1β high performance detection kit (part 62HIL1B2PEG).