Journal: PLoS ONE

Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

doi: 10.1371/journal.pone.0006209

Figure Lengend Snippet: Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).

Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG 3 anti- S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

Techniques: Blocking Assay, Incubation, Labeling

Journal: PLoS ONE

Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

doi: 10.1371/journal.pone.0006209

Figure Lengend Snippet: Adherent GM-MФs were incubated with unopsonized GFP- S. aureus RN6390. External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).

Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG 3 anti- S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

Techniques: Incubation, Labeling, Cytometry

Journal: eLife

Article Title: A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

doi: 10.7554/eLife.04491

Figure Lengend Snippet: ( A ) Sucrose gradient chromatograms (absorbance at 254 nm) for seven strains. Lines indicate the portion of the 30S peak that was collected and used for RNase H assay and EM analysis. ( B ) RNase assay cleavage detected on 2% agarose gel stained with ethidium bromide. Products are labeled on right. The anti-h21 oligo was used as a control, targeting a region of the rRNA that should be stable and inaccessible in all strains. ( C ) Quantitation of RNase H cleavage (average of three replicates, error bars represent standard deviation). For each lane, the intensity of intact 16S rRNA and cleavage products was measured using image quant. Cleavage products detected in the ‘no oligo’ lane were subtracted from other lanes for the same strain, to account for background cleavage that may have occurred before or during the cleavage assay. DOI: http://dx.doi.org/10.7554/eLife.04491.009

Article Snippet: Cleavage reactions were initiated on ice by adding 0.5 pmol 30S subunits (final concentration 33 nM) to 50 or 500 pmol (final concentration 3.3 or 33 µM) anti-PK or anti-h21 oligo (or buffer A for mock reactions) and 5U RNase H (New England Biolabs) (or buffer A for mock reactions).

Techniques: Rnase H Assay, Agarose Gel Electrophoresis, Staining, Labeling, Quantitation Assay, Standard Deviation, Cleavage Assay