Hek blue mtlr4 cells

NF-κB/secreted alkaline phosphatase (SEAP) reporter RAW264.7 cells (Novus Biologicals; catalog #NBP2-26260) are stably ransfected to express the SEAP reporter gene under the transcriptional control of an NF-κB response element. HEK-Blue-mTLR4 cells (Invivogen; catalog code hkb-mtlr4) are co-transfected with murine TLR4, MD-2, CD14, and inducible secreted embryonic alkaline phosphatase reporter gene. Cells were used at passages 1–3 after purchase. Validation data from the manufacturer were reviewed; further authentication (other than inclusion of positive and negative controls) was not performed. siRNA against murine Tlr4, Myd88, and Ticam1 were purchased from Thermo Fisher (catalog #AM16708). Mycoplasma testing was performed by PCR (Agilent Technologies; catalog #302106).

Histones (0–20 μg/mL, Sigma Cat# H9250), PAM3CSK4 (1 μg/mL, Invivogen, San Diego, CA, USA Cat# tlrl-pms), PAM2CSK4 (1 μg/mL, Invivogen, Cat# tlrl-pm2s-1), PHAD (1 μg/mL, Avanti Polar Lipids, Alabaster, AL, USA, Cat 699800) or CpG ODN2395 (1 μg/mL, Miltenyi Cat# 130-100-282) diluted in complete IMDM were added to naive T cells at the time of culture. For stimulation with NETs, NET preparations in MCDB 131 media or MCDB 131 media alone were similarly added at the start of the culture period. For experiments inhibiting histones/NETs, the inhibitor β-methyl-cellobioside sulfate (mCBS) (100 μg/mL) or PBS was added simultaneously with the addition of histones or NETs to cultures. Unless otherwise stated, in all experiments, histones/TLR agonists/NETs/mCBS were added at the start of the culture period and remained in the media for the duration of the cell culture. TLR4 activity of PHAD was confirmed using HEK-Blue™ mTLR4 Cells (Invivogen, Cat# hkb-mtlr4) as described by the manufacturer.

HEK-Blue-mTLR4 cells were purchased from InvivoGen (San Diego, CA, USA). They are stably transfected with mouse TLR4 (mTLR4), myeloid differentiation factor-2 (MD-2), cluster of differentiation-14 (CD14), and an inducible embryonic alkaline phosphatase (SEAP) reporter gene. The cells were cultured and maintained at 37°C with 5% CO₂ in complete Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1× HEK-Blue selection medium (InvivoGen, San Diego, CA), an antibiotic mixture for maintenance of HEK-Blue mTLR4 cell lines. The cells were seeded at 2.5 × 10⁴ cells/well in a 96-well plate and stimulated for 24 h with 100 μl undiluted sera from sham and burned mice. Salmonella enterica serotype Minnesota R595 lipid A (lot number MLA-24A; List Biological Laboratories, Inc.) was used as a positive control. The activation of NF-κB in HEK-Blue mTLR4 cells in response to TLR4 agonists was determined by a SEAP reporter assay at a wavelength of 620 nm using a VERSA max microplate reader (Molecular Devices, San Jose, CA). Endotoxin levels were assessed by Endosafe PTS chromogenic Limulus amebocyte lysate assay cartridges (Charles River, MA).

LPS was quantified using HEK-Blue-mTLR4 cells (Invivogen, San Diego, CA, USA), and plasma samples collected at sacrifice. Then, 180 µL of a cell suspension (1.4 × 10⁴ cells per mL in HEK-Blue Detection medium) (Invivogen) was added to 20 µL of each diluted (1:10) plasma sample. LPS (Sigma, St.Louis, MO, USA) was used as a 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.

Feces were collected at days 1, 3, and 6 post-AIEC challenge. Samples were weighed, and then homogenized (without marbles) in phosphate buffer saline (PBS) for 15 min before centrifugation at 12,000× g rpm, at 4 °C for 10 min. Lipocalin-2 was quantified with an ELISA kit (Biotechne, Minneapolis, MN, USA) in duplicate.
LPS activity in plasma samples was evaluated, in duplicate, using 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 (1:10) plasma sample. LPS (InvivoGen, San Diego, CA, USA) was used as the positive control and to calculate the standard range. Plates were incubated at 37 °C in 5% CO₂ for 24 h, and alkaline phosphatase activity was measured at 620 nm.