Total phospho human instantone elisa kit

PBMCs (10⁶ cells/mL) (4 volunteers) were incubated with or without LPS (10 µg/mL) and APG (50 µg/mL) for 2 h. Pellets from treated cells were collected for the measurement of NFκB phosphorylation using the NFκB p65 (Total/Phospho) Human InstantOne™ ELISA kit (Thermo Fisher Scientific). Absorbance was measured at 450 nm, and the phospho-NFκB p65 over total-NFκB p65 ratio was then calculated. Data are expressed in percentage relative to the control and compared to the positive control group (cells treated with LPS without extract).

A Total/Phopsho NF-κB p65 (Total/Phospho) Human InstantOne^™ ELISA Kit (Thermo Fisher Scientific), which is a sandwich ELISA-based kit, was used to measure the levels of total and phosphorylated NF-κB, as per the manufacturer’s instructions [30 (link)]. We used cell lysates of blood samples collected from individuals with LTBI (n = 10) and patients with active TB (n = 12) to measure the levels of total and phosphorylated NF-κB. The absorbance was read using an ELISA reader (Multiskan RC/MS/EX Microplate Reader, Thermo LabSystems, Thermo Fisher Scientific) at a wavelength of 450 nm.
To determine the levels of TNF-α, we used culture supernatants collected after blockade and stimulation with CFP + PMA. TNF-α levels were determined in samples from the patients’ samples (9 LTBI and 11 TB) using the BD OptEIA Human TNF Set Kit [28 (link)], as per the manufacturer’s instructions. The optical density (OD) was measured at 450 nm using an ELISA reader (Multiskan RC/MS/EX Microplate Reader, Thermo LabSystems, Thermo Fisher Scientific). The standard curve was calculated from the readings of different concentrations of recombinant cytokines provided in commercial kits (500 pg/mL, 250 pg/mL, 125 pg/mL, 65.5 pg/mL, 31.3 pg/mL, 15.6 pg/mL, 7.8 pg/mL, and 3.9 pg/mL). The actual concentrations were calculated from the standard curve (r² = 0.997) using the obtained OD values.

HT-29 and Caco2 cells were seeded with a cell number of 3 × 10⁵ per well in 24-well culture plates. After 24 h, cells were cultured and treated as described (Section 2.2). Cells were washed and collected to measure NF-κB phosphorylation using the NF-κB p65 (Total/Phospho) Human InstantOne™ ELISA Kit (Thermo Fisher Scientific, Waltham, MA, USA). An equal volume of the capture antibody reagent and the detection antibody reagent was prepared prior to the experiment. In each well, 50 μL of whole cell lysate, a cell lysis mix (negative control), and a positive control cell lysate were individually added onto the 96-well ELISA plate. Two types of capture antibody reagents, total-NF-κB p65 antibody, and phospho-NF-κB p65 antibody were separately incubated in each well of the pre-coated plate containing sample lysates (50 μL/well) for 1 h. The plate was washed before adding a detection reagent for 30 min and the detection reaction was sequentially inhibited by adding a stop solution (100 μL/well). The absorbance was immediately measured at 450 nm with a correction to 650 nm using an xMark™ microplate absorbance spectrophotometer (BioRad, Hercules, CA, USA). The phospho-NF-κB levels and the ratio of phospho-NF-κB p65 vs. total-NF-κB p65. Data were compared with the control or DSS-treated group. The assays were performed in triplicate.