Journal: RSC Advances

Article Title: Utilization of metabolic energy in treatment of ocular surface disorders: polyphosphate as an energy source for corneal epithelial cell proliferation

doi: 10.1039/c9ra04409d

Figure Lengend Snippet: Immunofluorescence and ESEM analysis of the outgrown cells, 3 d after initiating of the assays. In the first series the cell layers outgrown from the explants (ex) were stained with (A–C) DRAQ5 to identify the nuclei or double-stained with (D–F) rhodamine/phalloidin and DRAQ5 to express the complex multilayered arrangement of the cells migrated out from the explants; immunofluorescence light microscopy. The rim of the explant is highlighted (ex). In (G–I) higher magnification images of the epithelial cells showing the different degrees of differentiation of the cells; ESEM. In the controls (G) the cells are not continuously attached to each other with cell-to-cell junctions. In addition, a partial exfoliation of the cells is apparent. In contrast, cells migrated from the explants which had been incubated with hp-lysate (HPL) (H), or with polyP (I) smoothly attach to each other and form a homogeneous margin rim of microvilli on their cell surfaces. Surface areas with extensive microvilli decorations are marked (mic).

Article Snippet: Cell outgrowth and differentiation were analyzed after staining with DRAQ5 (nuclei) and rhodamine phalloidin (cytoskeleton), as well as by environmental scanning electron microscopy (ESEM).

Techniques: Immunofluorescence, Staining, Light Microscopy, Incubation

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Stiffness of pancreatic cancer cells is associated with increased invasive potential

doi: 10.1039/c6ib00135a

Figure Lengend Snippet: (A) Time series of images showing scratch wound invasion of pancreatic ductal cells through Matrigel. Wound confluence is the percentage of wound area covered by cells. Color legend: green is the wound area, blue shows wound confluence in the wound area, and grey represents the confluent cells outside of the wound area. Scale, 300 μm. (B) Schematic illustration showing the modified scratch wound assay. Cells are plated on a layer of thin Matrigel and invade into the thick 3D matrix of overlaid Matrigel that fills the scratch wound. The line plot shows quantification of wound confluence over time. The dotted line indicates the 72 h time point, which we use to compare wound confluence values for statistical significance. The bar plot represents wound confluence at the 72 h time point. Pairwise p-values are determined by a Student’s t-test. *p < 0.05. (C) Schematic illustration showing the transwell migration assay. Cells migrate through the 8 μm pores of a polycarbonate membrane in response to a chemoattractant on the opposite side of the membrane. Images of transwell migration assays showing DRAQ5-labeled nuclei of cells that migrate through the 8 μm pores of a polycarbonate membrane after 12 hours. Scale, 50 μm. Pairwise p-values are determined by a Student’s t-test. *p < 0.05. All error bars represent standard errors. The significance of pairwise comparisons between cell lines is shown in panels B and C by the initial(s) of the cell lines that are significantly different where H: HPDE, Hs: Hs766T, M: MIA PaCa-2, and P: PANC-1. For example, in panel B, HPDE is significantly different (*p < 0.05) from Hs766T (Hs) and PANC-1 (P).

Article Snippet: To image the nucleus, cells are stained with DRAQ5 (1:250 in 0.3% Triton X-100 in 1x PBS; ThermoFisher) at room temperature for 20 minutes.

Techniques: Modification, Scratch Wound Assay Assay, Transwell Migration Assay, Migration, Labeling

Journal: Parasitology Research

Article Title: Besnoitia besnoiti– driven endothelial host cell cycle alteration

doi: 10.1007/s00436-020-06744-x

Figure Lengend Snippet: Infection of BUVEC with B. besnoiti tachyzoites and effect of infection on host cell proliferation. Sub-confluent BUVEC were infected with B. besnoiti tachyzoites at an MOI 5:1 and analyzed after 24 hpi. a Quantification of tachyzoites numbers/parasitophorous vacuole during 30 h of infection. The mature structures were observed between 24 and 30 h p.i. After this time point, all parasites were released. b Illustration of non-infected control cells and B. besnoiti– infected BUVEC at 24 h p.i. c Host cell proliferation analyzed by estimating cell numbers of B. besnoiti –infected BUVEC and non-infected controls at 24 h p.i. No statistically significant differences were observed, but it was a hint of decreased number of cells at the B. besnoitia –infected monolayer. d Quantification of binucleated host cells in B. besnoiti –infected BUVEC and non-infected controls. e Illustration of DRAQ5 (vital DNA staining, red)-stained host cell nuclei in B. besnoiti – and T. gondii –infected BUVEC (24 h p.i.) via 3D holotomographic microscopy (note: binucleated phenotype in case of T. gondii –infected BUVEC)

Article Snippet: This was also tested via DRAQ5 (vital staining of nuclei)-based live cell 3D holotomographic microscopy (3D Nanolive®).

Techniques: Infection, Staining, Microscopy