Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Effects of CellTrace labeling on bacteria. ( A ) FC analysis of bacteria labeled with CellTrace. Gram-positive S. gordonii and Gram-negative P. gingivalis were labeled for 1 hour with CellTrace™ CFSE (green) and analyzed using FC. Graphs show CFSE staining expressed as the relative fluorescence intensity ( X -axis) and the relative number of events (counts, Y -axis). The data show three experiments carried out using independent biological replicates and a single population of unstained cells was used as a control. ( B ) Growth curve analysis of CellTrace CFSE-labeled planktonic cultures. Cells were pre-labeled for 1 hour, and growth was measured as optical density. Unlabeled cells were used for comparison. Graphs show mean ± SD of three independent biological replicates (except for P. gingivalis and P. micra where n = 2). Bar charts (inserts) show mean ± SD of CFU counts at experiment start and a later stage of growth for each bacterial species. Labeled and unlabeled cells are shown in green and black, respectively. ( C ) Fluorescence intensity of CellTrace™ CFSE-labeled planktonic cultures. Graphs showing mean ± SD of MFI values over time measured using FC. Two representative Gram positive ( S. gordonii and S. mutans ) and Gram negative ( P. gingivalis and V. parvula ) bacteria are shown.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Labeling, Bacteria, Staining, Fluorescence, Control, Comparison

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: CDSM visualization of SS biofilms labeled with CellTrace. Representative CDSM images of SS biofilms related to oral disease models. Biofilms were formed overnight using unlabeled or CellTrace (far red, yellow, CFSE, or violet)-labeled cells. Unlabeled biofilms were post-stained with CellTrace CFSE for comparison. The periodontitis model is represented by S. gordonii, A. odontolyticus, P. gingivalis, and P. micra, whereas the dental caries model is comprised of S. mutans , V. parvula, L. paracasei, and A. naeslundii . The scale bar (10 µm) applies to all panels.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Labeling, Staining, Comparison

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Comparison of surface coverage of SS biofilms labeled with CellTrace CFSE pre- or post-biofilm development ^a

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Comparison, Labeling

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Examination of biofilm structure and cell detachment from SS biofilms over time. Gram-positive ( S. gordonii and A. odontolyticus ) and Gram-negative ( P. gingivalis and V. parvula ) bacteria labeled with CellTrace CFSE (green) were used to create SS biofilms. Graphs (left panels) show percentages of CFSE-stained cells present in the starting inocula and biofilm supernatants. Supernatants were extracted and analyzed with FC on days 1, 2, and 3. The right panels show representative Z-plane CDSM images of SS biofilms after 4 hours (day 0) and 1, 2, and 3 days.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Bacteria, Labeling, Staining

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Assessment of CellTrace staining as a marker of bacterial viability in biofilms representative CDSM and CLSM images of overnight SS biofilms stained with CellTrace CFSE (green) or BacLight LIVE/DEAD stain. Biofilms of S. gordonii and P. gingivalis were maintained in PRNM (control) or treated with 70% ethanol or H 2 O 2 for 1 hour. The scale bar (10 µm) applies to all panels.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Staining, Marker, Control

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Visualization of MS biofilms representing a periodontitis model labeled by multiplexing CellTrace dyes. Representative CDSM Z-stack (left) and single-plane (right) images of overnight MS biofilms stained with CellTrace dyes. A unique CellTrace color was used to label four species ( P. gingivalis [red], S. gordonii [yellow], P. micra [green], and A. odontolyticus [violet]). Both multiplex (4-color compilation) and the color-segmented images are shown. The percentage coverage for each species within the biofilm models was calculated using a region of interest (ROI) within the delineated area in the single-plane image. Z-stack images were used to provide information regarding bacterial spatial location and relationships within the overall biofilm.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Labeling, Multiplexing, Staining, Multiplex Assay

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Visualization of MS biofilms representing a dental caries model labeled by multiplexing CellTrace dyes. Representative CDSM Z-stack (left) and single-plane (right) images of overnight MS biofilms stained with CellTrace dyes. A unique CellTrace color was used to label four species ( V. parvula [red], S. mutans [yellow], A. naeslundii [green], and L. paracasei [violet]). Both multiplex (4-color compilation) and the color segmented images are shown. The percentage coverage for each species within the biofilm models was calculated using a region of interest (ROI) within the delineated area in the single-plane image. Z-stack images were used to provide information regarding bacterial spatial location and relationships within the overall biofilm.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Labeling, Multiplexing, Staining, Multiplex Assay

Journal: Microbiology Spectrum

Article Title: A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro

doi: 10.1128/spectrum.00253-24

Figure Lengend Snippet: Assessment of CellTrace durability in MS biofilms. Representative CSDM and CLSM images of MS biofilms. Biofilms were created using the periodontitis model ( P. gingivalis [red], S. gordonii [yellow], P. micra [green], and A. odontolyticus [violet]) and visualized following overnight growth (day 1) and after 4 days through CDSM Z-stack images. Parallel, non-labeled biofilms were also grown, stained with BacLight LIVE/DEAD stain on day 4 and images using CLSM to assess viability. The scale bar (10 µm) applies to all panels.

Article Snippet: Histograms were then used to determine the median fluorescence intensity (MFI) of the whole population and the percentage of CellTrace CFSE-labeled cells within the population using BD Accuri C6Plus software.

Techniques: Labeling, Staining

Journal: Immunity & Ageing : I & A

Article Title: Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors

doi: 10.1186/s12979-020-00199-5

Figure Lengend Snippet: Suppression of PBMCs proliferation by myeloid subsets sorted from BM or BM-MDSCs. CellTrace-labelled PBMCs activated with anti-CD3 and anti-CD28 were cultured in the presence of 1:1 ratio of the myeloid populations, unsorted (UNS), mature (CD11b + ) or immature (CD11b − , BM-MDSCs). Immunosuppression of these populations was evaluated on activated T cells, (gated as CellTrace + /CD3 + ) normalized on the control without myeloid cells. ( n = 20 independent experiments for pediatric patients, n = 20 for elderly patients) a Suppression was calculated by CellTrace profile assessed as the reduction of the proliferating CD3 + cells (those contained within generation two onward) in the co-culture condition as compared to T cells cultured alone, expressed as percentage. b Suppression was assessed by T cell number calculated by analyzing the absolute number of proliferating CD3 + cells by TruCount™ tubes. In both cases proliferation data were normalized assuming as 100% the proliferation rate of CD3 + cells cultured without myeloid cells

Article Snippet: Proliferation of T cells was evaluated by assessing the signal of CellTrace on CD3 + cells, and considering as proliferating the cells present from generation 2 onwards, or by calculating the absolute number of CD3 + /CellTrace + cells in each sample by BD TruCount™ tubes (BD Biosciences).

Techniques: Cell Culture, Co-Culture Assay