HTRF Human and Mouse Total NF-κB Detection Kit, 10,000 Assay Points

Detection of total NFkB in various human/mouse cells

Human and murine cells in serum-deprived cell culture medium were plated at 40,000 cells per well in a 96-well plate and incubated for 24h at 37°C, 5% CO2. The phosphorylation state was induced by a 10 min stimulation time with 10 nM TNFalpha or 2 nM IL1beta. After stimulation, medium was removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into 384-well sv white microplate, and 4 µL of the HTRF total NFkB detection reagents were added. The HTRF signal was recorded after an overnight incubation.

 

HTRF assay vs WB using total-NFkB assay

Human HeLa cells were cultured for 48 followed by TNFalpha stimulation. Following lysis, soluble fractions were collected after centrifugation. Serial dilutions of the cell lysate were analyzed side-by-side by Western Blot and by HTRF. Results show that HTRF total-NFkB cellular assays is more sensitive than the Western Blot, as 1 000 cells are sufficient for minimal signal detection when using the HTRF total-NFkB assay while 4 000 are needed for a Western Blot signal.

 

TNFa dose-response on HeLa cells using phospho and total NFkB kit

HeLa cells were plated and cultured for 24h before being exposed to increasing concentrations of TNFalpha. Following cell lysis, 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the phospho-NFkB (Ser536) or total NFkB detection reagents were added. The HTRF signal was recorded after an overnight incubation. Stimulation with increasing concentration of TNFa induces phosphorylation of NFkB, while the total amount of protein, unphosphorylated and phosphorylated, remains stable.

 

 

Pharmacological response on Phospho and total NFkB of BAY 11-7085

Prolonged exposure to BAY 11-7085 induces a downregulation of the NFkB expression level, as shown by the decrease of the HTRF total NFKB signal. Phosphorylation of NFKB also declines with increasing BAY concentrations. Consequently, the normalization signal calculated from phospho- & total NFkB level remains stable. 40,000 cells of the U937 cell line were stimulated by increasing concentrations of BAY 11-7085 for a 3 hours, and co-stimulated for 10 min with 10 nM TNFalpha. Cells were lysed and transferred into a 384-well sv white microplate for detection of both HTRF phospho and total NFkB.