Journal: Frontiers in Cellular and Infection Microbiology
Article Title: Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII) and macrophages
Figure Lengend Snippet: BM and BP stimulation of various Toll-Like Receptors. To determine if BM and/or BP stimulated various TLRs, HEK293 cells expressing TLRs 4 and 5 and transfected with the firefly luciferase plasmid pNiFty2-Luc were stimulated with BM or BP at an MOI of 100:1. Un-stimulated cells served as negative controls. The TLR agonist served as positive controls. The cells were incubated with live bacteria for 6 h. The medium was then aspirated and replaced with fresh DMEM without antibiotics and incubated overnight at 37°C and 5% CO 2 . The TLR agonists remained with the cells for an overnight incubation. The following day, samples were analyzed to determine luciferase activity. Data is expressed as relative luciferase activity. Both BM and BP (B) stimulated TLR4 as effectively as the LPS agonist (A) . However, the flagellin from BP stimulated TLR5 (B) as we detected flagellin in the medium of B-infected cells but not BM (C) . Three experiments were performed in using triplicate samples and data is represented as mean ± SEM and * denotes significant differences of p
Article Snippet: TLR stimulation by BM or BP As described previously (Gentry et al., ), human embryonic kidney (HEK) 293 cells genetically manipulated to express human TLR4, or TLR5 (Invivogen, San Diego, CA) were cultured and maintained in DMEM (Cellgro Mediatech) supplemented with 10% FBS, 100 units/mL pencillin, 100 ug/mL streptomycin, 10 ug/mL basticidin, and 2 mM glutamine.
Techniques: Expressing, Transfection, Luciferase, Plasmid Preparation, Incubation, Activity Assay, Infection