Lipoteichoic acid

1,2-Dioleyloxy-3-dimethylaminopropane
(DODMA), 1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1′,2′,3′-triazole)triethylene
glycolmannose (PPM), cholesteryl hemisuccinate (CHEMS), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl) were purchased from Avanti Polar Lipids (Alabama,
U.S.A.). Lipoid S100 (soy phosphatidylcholine (SPC)) was a kind gift
from Lipoid GmbH (Ludwigshafen, Germany). Uranyless was purchased
from Electron Microscopy Sciences (U.S.A.). Methanol was purchased
from VWR International S.A.S. (Fontenay-sous- Bois, France). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), crystal violet 1%, Dulbecco Modified Eagle’s
medium (DMEM) high glucose, components of encapsulation buffer (EB)
(5 mM Tris-HCL, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM
MgCl2, 10 mM NaCl), α-hemolysin, lipase (from wheat germ, ∼0.1
U/mL), phosphate-buffered saline (PBS), fetal bovine serum (FBS),
penicillin-streptomycin, 5× trypsin, lipoteichoic acid (LTA),
and vancomycin were purchased from Sigma-Aldrich (Norway). AlexaFluor-594
labeled lipopolysaccharide (LPS) and AlexaFluor-594 labeled dextran
were purchased from Thermo Fisher Scientific. Tryptic soy broth (TSB)
and Luria broth agar (LA) were obtained from University Hospital of
North Norway (Tromsø, Norway). All the DNA sequences were purchased
from Integrated DNA Technologies (Belgium).^{15 (link)}

NMuMG cells were seeded at a density of 4×10⁵ cells/well in 6-well collagen-coated plates (#NCO3506, Corning, Corning, NY, USA) and incubated for 15 ± 1 h before experiments. After incubation, the cells were rinsed with sterile phosphate-buffered saline (PBS) and treated with LPS from Escherichia coli O111:B4 (#115K4029, Sigma-Aldrich, St. Louis, MO, USA) or lipoteichoic acid (LTA) from Bacillus subtilis (L3265, Sigma-Aldrich), which are outer membrane proteins of the Gram-negative and Gram-positive bacteria, respectively. Because our previous studies indicated significantly different responses in cultured mouse colonic, type-II alveolar, and pulmonary epithelial cells [16 (link),17 (link),18 (link)], LPS and LTA were diluted to 10 µg/mL in DMEM without FBS. NMuMG cells treated with LPS or LTA were incubated for 2 h at 37 °C in a 5% CO₂ atmosphere.