Journal: PLoS Neglected Tropical Diseases
Article Title: Comprehensive analysis of clinical Burkholderia pseudomallei isolates demonstrates conservation of unique lipid A structure and TLR4-dependent innate immune activation
Figure Lengend Snippet: Activation of NF-κB in HEK-Blue hTLR4 cells by B . pseudomallei K96243 cultured in different media. HEK-Blue hTLR4 cells were stimulated with heat-killed B . pseudomallei K96243 at 10 6 or 10 7 CFU/ml at 37°C in 5% CO 2 for 24 h. NF-κB activation was determined in the supernatant by a SEAP reporter assay with HEK-Blue detection. Mean ± SD of three independent experiment were illustrated. TSA; trypticase soy agar, LB; Luria-Bertani agar, Ash; Ashdown agar, BA; Blood agar, Mac; MacConkey agar, M9; M9 minimal medium agar.
Article Snippet: The bacteria were cultured on TSA (Oxoid, Hants, UK) for 16–18 h. Approximately 10 colonies were streaked onto agar plates and incubated for 16–18 h under one of the following conditions: (i) TSA (pH 7.4) at 25 o C, (ii) TSA (pH 7.4) at 37 o C, (iii) TSA (pH 7.4) at 42 o C, (iv) TSA (pH 4.5) at 37 o C, (v) TSA (pH 8.5) at 37 o C, (vi) TSA (pH 7.4) plus 2 mM H2 O2 (Merck, Darmstadt, Germany) at 37 o C, (vii) TSA (pH 7.4) plus 5 mM H2 O2 at 37 o C, (viii) TSA (pH 7.4) plus 50 mM NaNO2 (Sigma-Aldrich, MO, USA) at 37 o C, (ix) TSA (pH 7.4) plus 100 mM NaNO2 at 37 o C, (x) TSA (pH 7.4) plus 1 mM MgCl2 (Fisher Scientific, Leics, UK) at 37 o C, (xi) TSA (pH 7.4) plus 8 μM MgCl2 at 37 o C, (xii) Luria-Bertani agar (LB) (BD, MD, USA) (pH 7.4) at 37 o C, (xiii) Ashdown agar (pH 7.4) at 37 o C, (xiv) blood agar (Oxoid, Hants, UK) (pH 7.4) at 37 o C, (xv) MacConkey agar (Oxoid, Hants, UK) (pH 7.4) at 37 o C, and (xvi) M9 minimal medium agar (Sigma-Aldrich, MO, USA) (pH 7.4) at 37 o C. The bacteria were harvested using a 10 μl loop and further extracted for lipid A.
Techniques: Activation Assay, Cell Culture, Reporter Assay