Hek blue tlr4 cells

In Vitro Assays Using TLR4 Reporter Cell Lines. Murine or human TLR4 reporter cells (TLR4 HEK Blue cells, Invivogen, 2.5 × 10⁴ cells per well of a 96 well plate) were incubated with graded doses of the test compounds in DMEM with 10% FBS, 1 mM sodium pyruvate, 4 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Tested compounds were dissolved in dimethyl sulfoxide (DMSO) at 20 mM as a stock solution that were further serially diluted with DMSO and then added to the well (final DMSO concentration at 0.05%). The culture supernatants were harvested after a 20–24 h incubation period. SEAP activity in the culture supernatants was determined by a colorimetric assay, using QuantiBlue (Invivogen), with absorbance read at 630 nm. LPS (100 ng/mL, L4524, Sigma-Aldrich) was used as a positive control.

The CellSensor NFκB-bla human monocytic THP-1 cell line was purchased from Thermo Fisher Scientific (Waltham, MA). This cell line contains a stably integrated beta-lactamase reporter gene under the control of the nuclear factor kappa B (NF-κB) response element (https://tools.thermofisher.com/content/sfs/manuals/CellSensor_NFkBbla_THP1_man.pdf). NF-κB activation results in beta-lactamase production, which shifts the fluorescence emission of the beta-lactamase substrate (LiveBLAzerTM-FRET B/G (CCF4-AM), Thermofisher) to favor coumarin (460 nm emission) over fluorescein (530 nm emission). Murine or human TLR4 HEK Blue cells were purchased from Invivogen (San Diego, CA). These cell lines were stably cotransfected with human or mouse TLR4, MD-2, and CD14 coreceptor genes and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. QuantiBlue was purchased from Invivogen. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dipheyl tetrazolium bromide) was purchased from Acros Organics, and ovalbumin was purchased from Worthington Biochemical Corporation. For the HTS, LPS E. coli 0111:B4 (Sigma-Aldrich) was used, and LPS-EB Ultrapure (cat# tlrl-3pelps, Invivogen) was used in the validation and subsequent SAR studies. Wortmannin,^{33 (link)} a nonspecific, covalent inhibitor of PI3K, and MPLA were purchased from Invivogen.