Hek blue mtlr2 cells

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.

All cell lines were grown in 5% CO₂ at 37 °C, passaged at 80% confluence, and discarded after 20 passages. HT-29 (human colonic epithelial) cells and MODE-K (mouse duodenal epithelial) cells were a gift from Dr. Ali Ashkar (McMaster University). HT-29 cells were cultured in DMEM/F-12 with l-glutamine, HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). MODE-K cells were cultured in DMEM with l-glutamine, HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS. HEK-Blue mTLR2 cells were obtained from InvivoGen (San Diego, USA) and cultured in DMEM with l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin, and 10% FBS.
BMDCs were derived as previously described^{17 (link),46 (link)} using tibia and femurs from BALB/c mice. Cells were plated in 100 mm dishes at 10⁶/mL in 20 mL growth medium (day 0), refreshed on days 2 and 6, and harvested on day 7.

HEK-Blue mTLR2 cells (InvivoGen, San Diego, CA, USA) were used according to the manufacturer’s instruction to determine the serum concentrations of TLR2 ligands. After passaging according to the manufacturer’s instruction, a flat-bottom 96-well plate was loaded with 25,000 cells per well. Then, 20 μL of PBS, mouse serum, or serial dilutions, starting from 0.1 μg/mL, of Pam3CSK4, a synthetic triacylated lipopeptide, were added to each well. Cells were incubated for 6 h, spun down, and supernatant isolated. Twenty microliters of supernatant was then incubated with 180 μL of QUANTI-Blue solution (InvivoGen) for 15 min. Optical density at 620 nm was then measured using a Biotek Synergy H1M plate reader.