Journal: PLoS ONE

Article Title: Hydrogen Peroxide Produced by Oral Streptococci Induces Macrophage Cell Death

doi: 10.1371/journal.pone.0062563

Figure Lengend Snippet: Construction of S. oralis spxB deletion mutant. (A) Black arrow indicates the gene encoding pyruvate oxidase (SMSK23_0092 spxB). A targeted deletion mutant lacking this region was constructed by allelic exchange using the temperature-sensitive shuttle vector pSET4s. (B) S. oralis ATCC35037 wild-type (WT), spxB-deletion mutant (KO), or reverse mutant (Rev) was cultured in BHI broth or 5% RPMI1640 medium at 37°C for 18 h in a 5% CO 2 atmosphere. Concentrations of H 2 O 2 in culture supernatants were quantitatively determined using a hydrogen peroxide colorimetric detection kit. Data are shown as the mean ± SD of triplicate samples. *p

Article Snippet: Cell culture The human monocyte cell line THP-1 cells were purchased from RIKEN Bioresource Center and cultured in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS) (Invitrogen) (5% FBS RPMI1640), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a 5% CO2 atmosphere.

Techniques: Mutagenesis, Construct, Plasmid Preparation, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Analyses of Group III Secreted Phospholipase A2 Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis * Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis * S⃞

doi: 10.1074/jbc.M804628200

Figure Lengend Snippet: Foam cell formation from macrophages by PLA2G3, PLA2G5 and PLA2G10. A , LDL (2 mg/ml) was incubated with or without recombinant human PLA2G3 (sPLA 2 -III) and PLA2G5 (sPLA 2 -V) for 24 h at 37 °C. Then the NEFA released was quantified ( n = 2). B and C , J774 cells were cultured for 24 h in the presence or absence of LDL (200 μg/ml) that had been incubated for 24 h with or without PLA2G3 or PLA2G5. Then the cells were homogenized, and cholesteryl ester contents were quantified ( n = 2) ( B ). Replicate cells were fixed and stained with oil red O ( C ). Ac-LDL (50 μg/ml) was used as a positive control for foam cell formation. Magnification was ×200. D , peritoneal macrophages obtained from PLA2G3 Tg ( III-Tg ) mice and PLA2G10 Tg ( X-Tg ) mice as well as WT mice were placed in RPMI1640 containing 10% lipoprotein-deficient fetal bovine serum and were incubated with or without 200 μg/ml LDL or 50 μg/ml Ac-LDL. After 24 h, the cells were stained with oil red O (magnification, ×200). Magnified views of individual macrophages are shown in the inset. E , PGE 2 production by macrophages from PLA2G3 Tg ( left ) and PLA2G10 Tg ( right ) mice in comparison with WT mice; macrophages were incubated for 24 h in the presence (+) or absence (-) of 200 μg/ml LDL (mean ± S.E., n = 3–5; * , p

Article Snippet: Peritoneal cells isolated from mice were seeded in 2 × 106 cells/well in 12-well plates (Iwaki) for 2 h in RPMI1640 containing 10% lipoprotein-deficient fetal bovine serum (Sigma), and adherent cells were used as primary macrophages.

Techniques: Incubation, Recombinant, Cell Culture, Staining, Positive Control, Mouse Assay