Escherichia coli 0111b4 lps

Porphyromonas gingivalis strain W83 was grown in the absence or presence of cigarette smoke extract, as described previously [15 (link)]. In brief, Porphyromonas gingivalis cultures were grown to mid to late exponential phase in Gifu Anaerobe Medium (GAM, Nissui Pharmaceutical, Tokyo, Japan) or GAM-CSE under anaerobic conditions at 37°C in a Coy Laboratories anaerobic chamber (Coy Laboratory Products, Grass Lake, MI, USA). For Pg-CSE cultures, GAM was conditioned using standard reference cigarettes (Kentucky Tobacco Research and Development Centre, Lexington, KY, USA) and diluted to 4000 ng/ml nicotine equivalents prior to use [16 (link),17 (link)]. LPS extraction kit (iNtron Biotechnology, Kyunggi-do, Korea) was used for LPS extraction. LPS from Aggregatibacter actinomycetemcomitans strain Y4 (serotype B) was extracted and purified as previously described [18 (link)]. Escherichia coli (0111B4) LPS was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA) and included as an example of a non-periodontopathic bacterium for comparison. LPS was found to contain <0.001% nucleic acid and 0.7% protein by spectrophotometry and bicinchoninic acid protein assay, respectively [18 (link)].