Hek blue detection medium

Plasma myeloperoxidase (MPO) concentrations were measured using a commercial ELISA kit (Myeloperoxidase DuoSet ELISA, R&D Systems, Minneapolis, MN, USA). Triglycerides were measured in hepatic samples using a commercial kit (Cayman Chemical, Ann Arbor, MI, USA). Plasma lipopolysaccharide (LPS) activity was evaluated in HEK-Blue-mTLR4 cells (Invivogen, San Diego, CA, USA). Briefly, 180 µL of cell suspension (1.4 × 10⁴ cells per mL of HEK-Blue Detection medium) (Invivogen, San Diego, CA, USA) was added to 20 µL of each diluted (1:1000) plasma sample. LPS (Invivogen, San Diego, CA, USA) was used as positive control and standard range. Plates were incubated at 37 °C in 5% CO₂ for 24 h, and alkaline phosphatase activity was measured at 620 nm.

Specific-pathogen-free (SPF) female BALB/c mice aged 6–8 weeks old were purchased from the Experimental Animal Center of the Third Military Medical University, and were kept under pathogen-free conditions. HK-2 (human kidney 2) was purchased from the ATCC (American Type Culture Collection). HEK-Blue™-mTLR2 cells were purchased from InvivoGen (San Diego, CA, USA) and cells were grown in DMEM supplemented with 10% FBS, L-glutamine (2 mM), Normocin (100 μg/mL), penicillin (50 U/mL), streptomycin (50 g/mL) and passaged when reached 70% confluence. Cells were scraped and then resuspended in HEK-Blue™ Detection medium (InvivoGen, San Diego, CA, USA). The induction of SEAP was detected at 620 nm by a spectrophotometer.

Plasma samples were centrifuged and added to HEK-Blue mTLR4 cells in HEK-Blue detection medium (InvivoGen, San Diego, CA, USA). After 24 h of incubation, the color of secreted embryonic alkaline phosphatase (SEAP) released by the reporter cells was measured by a spectrophotometer at 620 nm (65 (link)).

For the Nod1 ligand bioactivity assay, HEK-blue™ mNOD1 293 cells (InvivoGen) were cultured according to manufacturer’s guidance. Cells were seeded 40,000 cells/well in a 96-well plate in 200 μL complete DMEM medium overnight. 2 μL mouse serum (65 °C 5 min heat inactivated) diluted with 18 μL PBS, or iE-DAP of different concentration in 20 μL PBS as standard was mixed with 180 μL HEK-Blue™ Detection medium (InvivoGen), respectively. Once samples and standard are prepared, all old media was discarded from 96-well plates, and then 200 μL of sample or standard solutions added. The plate was incubated in the 37 °C, CO₂ incubator for about 24 and absorbance read at 630 nm.
For the Nod2 ligand bioactivity assay, HEK293 cells stably expressing mouse Nod2 (InvivoGen) were cultured according to manufacturer’s guidance. Cells were seeded at a density of 3 × 10⁵ cells per well in a 24-well plate before transfection. Cells were transfected with 500 ng NF-κB-luc, 50 ng Renilla plasmid and 5 μL serum with Lipofectamine 2000, following the previous procedure.^{43 (link)} Luciferase expression was measured 24 h after transfection using a Luciferase Assay System (Promega, E2920) according to the manufacturer’s instructions.

An equal number (5–6×10⁶) of PU/ER(T) or HEK293 cells were electroporated with pGL3-KLF4 promoter constructs and pRLTK using Amaxa Electroporation kit. 36 h after transfection, cells were washed with cold PBS and lysed in 100 μl of Promega cell lysis buffer. Cell extracts were assayed for luciferase activity using a Promega dual luciferase assay system. Similarly, HEK293 cells were electroporated with pcpgf-KLF4 and grown in Invivogen HEK-Blue detection medium. mSEAP reporter activity was measured by absorbance of the growth medium at 630 nm.

HEK-Blue hTLR7 and hTLR8 cells (InvivoGen) were cultured in DMEM supplemented with 10% FBS, blasticidin, zeocin and normocin (InvivoGen). The cells (2.5 × 10⁴/well) were seeded into 96-well plates and then treated with the compounds in HEK-Blue Detection medium (InvivoGen) for 14 h. The activation of TLR7 and TLR8 was assessed by a microplate reader (BioTek, Winooski, VT, USA).