Macrophage activating lipopeptide malp 2

Tryptic soy broth (TSB), N-acetylmuramic acid, TRI Reagent, l-benzoyl-Arg-pNA (l-BApNA), and Tos-Gly-Pro-Lys-pNA (Tos-GPK-pNA) substrates, gentamicin, LPS from Escherichia coli O111:B4, lipoteichoic acid (LTA), and carboxymethylcelullose were from Sigma-Aldrich. Yeast extract, l-cysteine, hemin, and bovine serum albumin (BSA) were from BioShop Canada, Inc. Dulbecco’s phosphate-buffered saline without Ca²⁺ and Mg²⁺ (PBS), fetal bovine serum (FBS), RPMI 1640, the BCA protein assay kit, reducing sample buffer, Hoechst 33342 stain, eosin, and decalcifier were from Thermo Fisher Scientific. Menadione was from ICN Biomedicals, KYT-1 and KYT-36 from PeptaNova, and saponin from Serva Electrophoresis GmbH. Toll-like receptor agonists were obtained as follows: Pam₃CSK_4, flagellin, and R848 from Enzo Life Sciences, macrophage-activating lipopeptide (MALP-2) from Imgenex, CpG ODN 2006 from Hycult Biotech, and human recombinant IL-1β from BioLegend. Their purity was estimated for CpG 85% (high-performance liquid chromatograph [HPLC]), flagellin 95% (SDS-PAGE), LPS 80%, MALP-2 95% (HPLC), and LTA 97%, according to the manufacturer’s statement. All agonists, except LPS, were endotoxin free.