AlphaLISA SureFire Biotin-Free Human Total NLRP3 Detection Kit, 500 Assay Points

Activation of NFkB results in increased levels of NLRP3 in TLR4 overexpressing cells

THP-1 TLR4 overexpressing (OE) cells (InvivoGen, thpd-mctlr4) were seeded at 100,000 cells/well in a 96 well plate and treated with 1 µg/mL LPS for the indicated time points.

After treatment, cells were spun down and cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NLRP3 Total and NFkB Phospho (Ser536) levels were evaluated using respective AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 10,000 cells for NLRP3 or 1,000 cells for NFkB) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, LPS stimuli induced NFkB activation/phosphorylation which resulted in a modest increase of NLRP3 expression levels.

NLRP3 inflammasome activation in TLR4 overexpressing cells

THP-1 TLR4 overexpressing cells (InvivoGen, thpd-mctlr4) were seeded at 150,000 cells/well in a 96 well plate and left untreated or treated with LPS (500 ng/mL, 4h), Nigericin (10 µM, 30 minutes) or LPS + Nigericin (LPS 4h + Nig 30 minutes).

After treatment, cells were spun down, supernatant harvested and cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NLRP3 Total levels were evaluated in the cell lysate using the AlphaLISA SureFire Biotin Free assay, while the presence of IL-1b was evaluated in the collected supernatants. For the detection step, 10 µL of cell lysate (approximately 15,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, priming with LPS induced NLRP3 expression followed by IL-1b secretion which demonstrates activation of the inflammasome pathway.

Detection of NLRP3 in human PBMCs

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll Plaque Plus (Merck GE17-1440-02) and lysed at 6 x 10⁶ cells/mL with Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and assayed for NLRP3 Total using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells/datapoint is indicated.

Differential expression of NLRP3 Total (Human)

Adherent cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO_2. Suspension cells were harvested, washed, and seeded in a 96-well plate (100,000 cells/well) in HBSS.

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NLRP3 Total levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 10,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, expression of NLRP3 was only observed in THP-1 and U937 cells.

Assay sensitivity

Cell lysate was prepared from THP-1 cells lysed at 2 x 10⁶ cells/mL in lysis buffer for 10 minutes at RT with shaking. Lysate was serially diluted in Lysis Buffer and NLRP3 Total levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. This assay can detect NLRP3 expression in less than 200 cells/datapoint.