Journal: Scientific Reports

Article Title: Nucleoside-Diphosphate-Kinase of P. gingivalis is Secreted from Epithelial Cells In the Absence of a Leader Sequence Through a Pannexin-1 Interactome

doi: 10.1038/srep37643

Figure Lengend Snippet: NDK was visualized by transmission electron microscopy using immunogold labelling and rabbit anti- P. gingivalis NDK antibody. NDK (blue arrows) is seen on the P. gingivalis bacterial surface and in the host cytoplasm independently of the bacteria ( A,B ). An enlarged image of the boxed area is shown to the right ( A ). P. gingivalis with no primary antibody incubation ( C ), ndk- deficient mutant strain, ΔNDK ( D ) and GECs without infection ( E ) with both primary and secondary antibody incubations were used as controls. The black arrows point to non-specific background level of gold labelling staining in the control samples. Bar represents 1 μm.

Article Snippet: Coverslips with fixed cells were mounted onto Corning glass microscopy slides using VectaShield mounting medium containing DAPI (Vector Laboratories).

Techniques: Transmission Assay, Electron Microscopy, Bacteria, Incubation, Mutagenesis, Infection, Staining, Control

Journal: Molecular Microbiology

Article Title: A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface

doi: 10.1111/mmi.14898

Figure Lengend Snippet: S .Tm doublets promote epithelial cell invasion under virulence‐inducing conditions. (a–f) Variation in doublet frequency, motility, and expression of TTSS‐1 across different time‐points post subculture (p.sc.), under narrow (a–c) or broad (d–f) induction conditions, quantified by single‐cell microscopy. Gray shading denotes the late exponential phase–early stationary phase transition (time‐points used in subsequent experiments). Each panel shows data as mean ± SD of three experiments (very low SD not visible for some points). (a) Quantification of doublets in S .Tm wt cultures grown ON, sub‐cultured 1:100 in LB medium (narrow induction condition), and incubated 0–24 h before imaging. Data expressed as a fraction of total population (blue curve, left y‐axis). The growth curve is represented by a sigmoidal fit of OD 600 measurements (gray curve, right y‐axis; fit excludes the 24 h time‐point). (b) Fraction of motile bacteria (>5 μm/s) observed under conditions as in a, generated by single‐particle tracking. (c) The SPI‐1 reporter strain S .Tm/p sicA ‐GFP was grown as in a and the frequency of GFP‐expressing bacteria was quantified by microscopy. (d–f) Similar data as in a‐c but acquired for S .Tm cultures grown under the broad induction condition (12 h ON followed by 1:20 subculture in LB/0.3 M NaCl). (g) Example DIC images of a singlet and a doublet in the inoculum used for infections. (h) Representative time series of HeLa cells infected with an S .Tm/p rpsM ‐GFPmut2 constitutive reporter strain. Cells in greyscale, bacteria (GFP) in green. Arrows denote examples of invading singlet (white arrow) and doublet S .Tm (yellow arrow), the latter subsequently dividing into two daughter cells. Entry ruffles are delimited by dotted lines (right‐most panel). The time indicated in minutes. (i and j) Quantification of the frequency of S .Tm doublets in the inoculum (x‐axis) vs. in the corresponding ruffle‐inducing population in HeLa cells during the first 30 min of co‐incubation (y‐axis). The black dotted line illustrates a theoretical 1:1 ratio ( k = 1). For i and j, each graph shows data for 3–6 h subcultures used as inocula, pooled from three independent experiments (total n = 12 infections). Data from different subcultures are shown as filled circles and linear regression as dashed lines for the narrow (i; k = 2.2) and broad (j; k = 2.6) induction condition, respectively. Scale bars in all panels: 3 μm

Article Snippet: Cells for fixation were grown in either multi‐well glass‐bottomed plates or on glass microscopy slides with detachable 8‐well walls (#1.5H; MatTek) for short‐ or long‐term storage, respectively.

Techniques: Expressing, Microscopy, Sublimation, Cell Culture, Incubation, Imaging, Bacteria, Generated, Single-particle Tracking, Infection

Journal: Molecular Microbiology

Article Title: A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface

doi: 10.1111/mmi.14898

Figure Lengend Snippet: Singlets and doublets exhibit different search patterns atop epithelial cell layers. (a and b) Representative micrographs of a murine enteroid‐derived monolayer, imaged using (a) DIC and (b) fluorescence microscopy. Monolayers were seeded and grown for 3 days, fixed, and stained with DAPI (blue) and Alexa Fluor 488 Phalloidin (green). (c) Stereotypic hexagonal grid representation of the monolayer, used in later analyses. Hexagons have an area corresponding to the experimentally determined mean for individual cells within the monolayer (112.9 μm 2 ; n = 590 cells). (d) Representative examples of experimentally determined near‐surface swim paths (black arrows) for S .Tm ΔinvG singlets (left) and doublets (right), superimposed on top of the hexagonal grid representation. Traversed hexagons are highlighted in blue. Data from time‐lapse microscopy (frame interval: 100 ms) of murine enteroid‐derived monolayer co‐incubations. (e) Conceptual illustration for track elongation by repetition, used to create extrapolated swim paths from the time‐restricted experimental data in d. In each iteration, a track (black arrow) is copied (gray arrow), moved to start at the last point of the original track, and rotated so that the direction of the vector formed by its first two points matches the last two points of the original (forming a 0° angle). Tracks were repeated 50 times, forming overlapping paths (thus mitigating differences in track length). (f) Representative examples of extrapolated swim paths for singlets and doublets, generated by elongation of experimentally determined swim paths, as in e. Traversed hexagons are highlighted in blue. (g) Quantification of hexagons traversed by all extrapolated swim paths for S .Tm ΔinvG singlets and doublets in the group of fastest swimmers (inclusion criteria: speed ≥15 μm/s, tracked for ≥1.5 s; total n = 84). Filled dots show individual measurements, lines represent medians. Data pooled from three independent experiments. Statistical analyses via Mann–Whitney U test (*: p < 0.05). The black dot in panels d–f indicates the path origin. Scale bars in panels a–c: 10 μm, in panels d, f: 100 μm

Article Snippet: Cells for fixation were grown in either multi‐well glass‐bottomed plates or on glass microscopy slides with detachable 8‐well walls (#1.5H; MatTek) for short‐ or long‐term storage, respectively.

Techniques: Derivative Assay, Fluorescence, Microscopy, Staining, Time-lapse Microscopy, Plasmid Preparation, Generated, MANN-WHITNEY

Journal: Scientific Reports

Article Title: An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip

doi: 10.1038/s41598-021-96609-9

Figure Lengend Snippet: Experimental setup of high-throughput droplet-based cytotoxicity platform. ( A ) Experimental schematics showing cytotoxicity platform that combines (i) droplet generation and cell pairing using microfluidics, (ii) droplet immobilization for real-time microscopy, (iii) automated image analysis using custom-made MATLAB script to allow unbiased and high throughput detection of cytotoxic events. Stained NK cells and K562 cells were loaded into the chip using 200 µL pipette tips and encapsulated into droplets using a 3-inlet microfluidic chip. The viability dyes were included within the cell medium. The immobilized droplets were incubated in a stage top incubator set at 5% CO 2 and 37 °C. Image acquisition was performed at every hour interval for 10 h. ( B ) The three-inlet microfluidic device with flow-focusing junction to generate droplets. ( C ) A qualitative test of the observation chamber was performed by monitoring droplets movement in the chamber under the microscope for 10 h.

Article Snippet: Glass microscopy slides (76 × 26 × 1 mm; Corning) were used as top and bottom covers (76 × 26 × 1 mm).

Techniques: High Throughput Screening Assay, Microscopy, Staining, Transferring, Incubation