rabbit anti streptococcus group d polyclonal antibody  (American Research Products)

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A) Quantification of intracellular replicating (eFluor - ) and non-replicating (eFluor + ) E. faecalis strains from macrophage lysates at 2, 6, and 20 hpi using flow cytometry. WT OG1RF fixed with 4% PFA prior to infection (Fixed WT) was included as non-proliferating controls. Representative histograms from n = 2-4 are shown. (B) Proportion (%) of intracellular replicating E. faecalis (eFluor - ) population from WT and mutant E. faecalis -infected macrophage lysates. Bars represent mean ± SD of n = 4, except fixed WT (n = 2). Statistical significance of each strain against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. Only comparisons with p < 0.05 are annotated. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. (C) Representative confocal microscopy images of RAW264.7 macrophages infected by E. faecalis strains pre-stained with eFluor 670 (magenta) at 20 hpi from n = 3. Samples were fixed and post-stained for Enterococcus -specific Group D antigen (green), double-stranded DNA (dsDNA; blue) and F-actin (white).

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Flow Cytometry, Infection, Mutagenesis, Confocal Microscopy, Staining

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A) LDH cytotoxicity quantification of culture supernatants from E. faecalis -infected macrophages at 2, 6 and 20 hpi using the antibiotic protection assay. Bars represent mean ± SD from n = 5. Statistical significance against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. A subset of this data comparing macrophage cytotoxicity from WT infection at 2, 6 and 20 hpi is shown in . (B-C) Quantification of extracellular egressed bacteria from culture supernatants of RAW264.7 macrophages infected with (B) fsrABDC/gelE deletion mutants and (C) gelE -complemented OG1RF strains at 6 to 12 hpi. Each data point represents mean ± SD of n = 3-4. At each timepoint, statistical significance against WT was assessed using two-way ANOVA with Dunnett’s multiple comparisons test. LOD = limit of detection. (D) Representative Z-projections of WT- and Δ gelE -infected RAW264.7 macrophages at 6, 9 and 12 hpi from (B) , captured with confocal microscopy and stained for Enterococcus specific Group D antigen (yellow), dsDNA (cyan), and F-actin (magenta) (n = 2). Orthogonal Z-axis projections along the blue and red lines are shown in the adjacent colored boxes. White and yellow arrows indicate matched top view and Z-axis projections of selected macrophages with dense intracellular E. faecalis . For all graphs, only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Infection, Bacteria, Confocal Microscopy, Staining

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A-B) Total CFU from wound homogenates of (A) mono-infection or (B) competitive infection at 1, 3, and 5 dpi. Median of 3-5 animals per group from one independent experiment is shown. Dotted lines show inoculum CFU for mono-infection. (C) Flow cytometry analysis of intracellular E. faecalis (stained for Group D antigen) in various immune cells at 5 dpi. Median from 6-9 animals per group from 2 independent experiments is shown. (D-E) Quantification of (D) intracellular CFU at 1, 3 and 5 dpi or (E) total CFU at 5 dpi from enzymatically dissociated wounds. Median from 4-5 animals per group from one independent experiment is shown. For A-D, only comparisons with p < 0.05 are annotated. At each timepoint, statistical significance between infection groups was assessed using Mann-Whitney test, except for C, which was assessed using Kruskal-Wallis test with Dunn's multiple comparisons test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n.s. = not significant.

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Infection, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Infection and Immunity

Article Title: Enterococcus faecalis persists and replicates intracellularly within neutrophils

doi: 10.1128/iai.00364-25

Figure Lengend Snippet: Intracellular clusters of E. faecalis within neutrophils in the murine wound bed at 24 h p.i. Mice were wounded and infected with 10 6 CFU of E. faecalis OG1RF ( A–C ) or OG1RF pDasher ( D, E ) for 24 h. ( A–C ) Wounds were excised, fixed, and embedded before being cryosectioned, mounted on slides, and stained for Streptococcal Group D antigen to mark E. faecalis bacteria (yellow) and DAPI for DNA (cyan). Sections were imaged at 63× on a confocal microscope. ( A ) Depicts a tile scan of a portion of the entire wound bed, with tissue and surface sides indicated. The white inset box is shown in ( B ) as a single slice from the z stack with ortho view and in ( C ) as a max intensity projection, with additional zoomed-in angled orange and red insets of IMARIS 3D reconstructions. ( A–C ) Are from a single experiment and mouse, representative of N = 6 mice from three independent experiments. ( D–E ) Wounds were excised, and then cells were dissociated from this tissue via liberase reagent. Cells were then stained and imaged via a BD LSRFortessa X-20 Cell Analyzer. ( D ) Displays the relative proportion of (Ly6G high , CD11b + ) neutrophils compared to (Ly6G low , F4/80 + , CD11b + ) macrophages in the CD45 + immune cells analyzed. ( E ) Further displays the proportion of neutrophils that were EGFP (i.e., E. faecalis OG1RF pDasher-positive). ( D, E ) Depict mean + SEM from N = 6 mice across three independent experiments.

Article Snippet: Sections were then blocked using blocking serum (PBS plus 5% FBS, 5% BSA, 0.05% Triton) for 1 h. Both were then incubated overnight at 4 °C with polyclonal rabbit antibodies against Streptococcus Group D antigen (1:500 dilution from whole serum aliquot) (#12-6231D, American Research Products) in either 1% BSA (coverslips) or antibody diluent solution (PBS plus 1% FBS, 1% BSA, 0.05% Triton).

Techniques: Infection, Staining, Bacteria, Microscopy

Journal: Infection and Immunity

Article Title: Enterococcus faecalis persists and replicates intracellularly within neutrophils

doi: 10.1128/iai.00364-25

Figure Lengend Snippet: Engulfment and intracellular persistence of E. faecalis within murine neutrophils to 24 h p.i. Murine neutrophils were pre-stained with CellTracker Blue (cytoplasm, magenta), then infected with E. faecalis OG1RF for 6 h, followed by gentamicin exclusion of extracellular bacteria. At ( A, C ) 6 or ( B, D ) 24 h p.i., cells were fixed with 4% PFA and stained for Streptococcal Group D antigen ( E. faecalis bacteria, yellow) before being imaged at 63× on a confocal microscope. ( C–D ) Images from (four per condition, minimum 50 cells total) were blindly quantified to determine the total number of bacteria, neutrophils, and infected neutrophils. ( A–B ) Depict a single experiment, representative of n = 4 independent experiments. ( C–D ) Depict mean ± SEM, from the same n = 4 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; * denotes P < 0.05, **P < 0.01.

Article Snippet: Sections were then blocked using blocking serum (PBS plus 5% FBS, 5% BSA, 0.05% Triton) for 1 h. Both were then incubated overnight at 4 °C with polyclonal rabbit antibodies against Streptococcus Group D antigen (1:500 dilution from whole serum aliquot) (#12-6231D, American Research Products) in either 1% BSA (coverslips) or antibody diluent solution (PBS plus 1% FBS, 1% BSA, 0.05% Triton).

Techniques: Staining, Infection, Bacteria, Microscopy

Journal: Infection and Immunity

Article Title: Enterococcus faecalis persists and replicates intracellularly within neutrophils

doi: 10.1128/iai.00364-25

Figure Lengend Snippet: Intracellular replication of E. faecalis within murine neutrophils, between 6 and 18 h p.i. Murine neutrophils were pre-stained with CellTracker Blue (cytoplasm, magenta), then infected with E. faecalis OG1RF for 6 h, followed by gentamicin exclusion of extracellular bacteria. At ( A ) 7 h or ( B ) 18 h p.i., RADA (fluorescent D-amino acid incorporated into bacterial cell walls during synthesis, cyan) was added into the high-dose gentamicin media. At 24 h p.i., cells were fixed with PFA and stained for Streptococcal Group D antigen ( E. faecalis bacteria, yellow) before being imaged at 63× on a confocal microscope. Scale bar in insets depicts 10 mm. Images depict a single experiment, representative of n = 4 independent experiments.

Article Snippet: Sections were then blocked using blocking serum (PBS plus 5% FBS, 5% BSA, 0.05% Triton) for 1 h. Both were then incubated overnight at 4 °C with polyclonal rabbit antibodies against Streptococcus Group D antigen (1:500 dilution from whole serum aliquot) (#12-6231D, American Research Products) in either 1% BSA (coverslips) or antibody diluent solution (PBS plus 1% FBS, 1% BSA, 0.05% Triton).

Techniques: Staining, Infection, Bacteria, Microscopy