Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: APL Bioengineering

Article Title: A high throughput cell stretch device for investigating mechanobiology in vitro

doi: 10.1063/5.0206852

Figure Lengend Snippet: Stretch plate automation compatibility. (a) The workflow graphic shows the 192-well linear stretch plate with an aluminum plate holder that was constructed to support the stretch plate and allow automation gripping mechanisms to firmly clasp the plate. The combined plate and holder facilitate the use of automated platforms such as the GNF Systems Plate Washer-Dispenser for collagen coating and cell dispensing into stretch plates, and Opera Phenix High-Content Screening System to image cells. (b) Images show Hoechst-stained nuclei from human primary chondrocytes dispensed into collagen coated stretch plates and after an overnight attachment. The left image (192w stretch plate) is a full plate view composite of stitched and z-stack images from each of 192 wells. Middle panels (well G2 inset) show brightfield and fluorescent images from a representative stitched single well, and right panels (field 1 inset) show single 10× images from the stitched single well series for a higher magnification view of Hoechst-stained nuclei and brightfield imaging of chondrocytes. Images were reproduced with permission from GNF Systems and Revvity.

Article Snippet: Cells were then resuspended in Chondrocyte Growth Medium (Lonza Kit CC3216: CC3217 + CC4409) for cell culture.

Techniques: Construct, High Content Screening, Staining, Imaging

Journal: APL Bioengineering

Article Title: A high throughput cell stretch device for investigating mechanobiology in vitro

doi: 10.1063/5.0206852

Figure Lengend Snippet: Live cell image analysis of EthD-1 with stretch. Human primary chondrocytes were subjected to 20% cyclic linear stretch at 1 Hz for 2 h and then co-stained with the nuclei stain Hoechst and the cell death and damage marker ethidium homodimer-1 (EthD-1). Full plates were scanned and imaged using the Opera Phenix High-Content Screening System for image stitching and z-stacking to capture the total cell population. (a) Representative images of cells stained with Calcein-AM from static and stretched plates are shown. (b) Total cell number in each of 192-wells across the static and stretch plates was visualized using cells positively stained with Hoechst and plotted in plate view from a representative plate using TIBCO Spotfire software. A blue-shaded gradient color scale representing the total cell number from 0 to 30 000 is used. (c) Percent cells positively stained with EthD-1 were plotted in plate view from a representative plate to visualize percentage of damaged cells per well across the stretch plate under static and stretch conditions. A red-shaded gradient color scale representing percent positive cells from 0 to 10 is used. (d) The total number of nuclei was counted, and plate average and standard error of the mean were plotted for four static and four stretch plates. (e) Percent positive cells were quantified, and plate average and standard error of the mean were plotted for four static and four stretch plates. Significance was performed in GraphPad Prism 9, using an unpaired t-test with Welch correction and Holm-Šídák method and p-value < 0.05. No significant differences were determined for (c). (Hoechst p-value = 0.787) or D. (EthD-1 p-value = 0.086).

Article Snippet: Cells were then resuspended in Chondrocyte Growth Medium (Lonza Kit CC3216: CC3217 + CC4409) for cell culture.

Techniques: Staining, Marker, High Content Screening, Software

Journal: APL Bioengineering

Article Title: A high throughput cell stretch device for investigating mechanobiology in vitro

doi: 10.1063/5.0206852

Figure Lengend Snippet: Stretch-stimulated gene signature. RASL sequencing of cellular landmark and chondrocyte genes was performed to identify stretch responsive genes. (a) Volcano plot shows differentially expressed transcripts with stretch compared to static conditions, from human primary chondrocytes subjected to 20% cyclic linear stretch at 1 Hz for 2 h. Stretch genes were defined as above thresholds set to −log10padj > 50 and |log2FC| > 0.5. (b) Gene expression plotted in plate view using TIBCO Spotfire software is shown as average reads per million (RPM) for each well across the static and stretch plates, for either the up-regulated gene set (up-regulated stretch response, left) or the down-regulated gene set (down-regulated stretch response, right). Average RPM values are visualized using color and shape, scaling shown in respective graph legends. (c) Cell pathway and transcription factor enrichment based on the identified stretch responsive genes were determined using Metascape (metascape.org) online software.

Article Snippet: Cells were then resuspended in Chondrocyte Growth Medium (Lonza Kit CC3216: CC3217 + CC4409) for cell culture.

Techniques: Sequencing, Gene Expression, Software

Journal: APL Bioengineering

Article Title: A high throughput cell stretch device for investigating mechanobiology in vitro

doi: 10.1063/5.0206852

Figure Lengend Snippet: Stretch responsive genes. RASL sequencing of cellular landmark and chondrocyte genes was performed for chondrocytes subjected 20% cyclic linear stretch at 1 Hz for 2 h. To identify stretch responsive genes, differentially expressed transcripts with stretch compared to static conditions were defined as above thresholds set to −log10padj > 50 and |log2FC| > 0.5. There were 29 stretch responsive genes identified (12 up-regulated and 17 down-regulated).

Article Snippet: Cells were then resuspended in Chondrocyte Growth Medium (Lonza Kit CC3216: CC3217 + CC4409) for cell culture.

Techniques: Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Differential gene expression of human chondrocytes cultured under short-term altered gravity conditions during parabolic flight maneuvers

doi: 10.1186/s12964-015-0095-9

Figure Lengend Snippet: Quantitative real-time PCR analysis of chondrocytes exposed to the Vibraplex device. The Vibraplex device provides vibration profiles corresponding to those occurring during parabolic flight and allows the isolated analysis of their effects on cultivated cells. In all diagrams, the x-axis represents the experiment conditions and the y-axis the relative gene expression in % of control. Results are given as mean value ± SD. Significant changes ( p < 0.05) are indicated by brackets above the bars. The analyzed genes were A : CTGF ; B : IL6 ; C : CAV2 ; D : IL8 ; E : EGF ; F : PRKAA ; G : VEGFA ; H : PRKCA ; I : VIL2 ; J : VEGFD ; K : FGF17 ; L : IL15.

Article Snippet: Commercially available human chondrocytes (Provitro®, Berlin, Germany) were cultured in Chondrocyte Growth Medium basal (CGM, Provitro®, Berlin, Germany) supplemented with 10% fetal calf serum (Provitro®, Berlin, Germany), 100 IU penicillin/mL and 100 μ g streptomycin/mL (Provitro®, Berlin, Germany).

Techniques: Real-time Polymerase Chain Reaction, Isolation, Gene Expression, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Differential gene expression of human chondrocytes cultured under short-term altered gravity conditions during parabolic flight maneuvers

doi: 10.1186/s12964-015-0095-9

Figure Lengend Snippet: STRING analysis of the cluster 6 from the parabolic flight experiment. Chondrocytes were fixed after parabola 1 and 31 during a parabolic flight. In parallel, corresponding 1 g control samples were prepared. K-mean clustering of the resulting microarray data revealed 6 clusters of differentially expressed genes. Clusters 1–5 revealed mostly unspecific transcriptionally active genes, while cluster 6 showed a strong dominance by anti-apoptotic and cell-proliferative transcripts. Possible interactions of the corresponding proteins were visualized using the STRING software and genes involved in anti-apoptosis and cell proliferation were highlighted with white and black circles, respectively.

Article Snippet: Commercially available human chondrocytes (Provitro®, Berlin, Germany) were cultured in Chondrocyte Growth Medium basal (CGM, Provitro®, Berlin, Germany) supplemented with 10% fetal calf serum (Provitro®, Berlin, Germany), 100 IU penicillin/mL and 100 μ g streptomycin/mL (Provitro®, Berlin, Germany).

Techniques: Control, Microarray, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Differential gene expression of human chondrocytes cultured under short-term altered gravity conditions during parabolic flight maneuvers

doi: 10.1186/s12964-015-0095-9

Figure Lengend Snippet: Quantitative real-time PCR analysis of chondrocytes after exposure to parabolic flight. Chondrocytes were taken on a parabolic flight and fixated at two different timepoints. Subsequently, qPCR analyses were performed on these samples and the corresponding 1 g controls. In all diagrams, the x-axis represents the experiment conditions and the y-axis the relative gene expression in % of control. Results are given as mean value ± SD. Significant changes ( p < 0.05) are indicated by brackets above the bars. The analyzed genes were A : CCNA2 ; B : CD44 ; C : IL6 ; D : IL8 ; E : VCAM1 ; F : EDN1 ; G : TNFA ; H : FGF9 . 1P: fixation after first parabola; 31P: fixation after 31st parabola.

Article Snippet: Commercially available human chondrocytes (Provitro®, Berlin, Germany) were cultured in Chondrocyte Growth Medium basal (CGM, Provitro®, Berlin, Germany) supplemented with 10% fetal calf serum (Provitro®, Berlin, Germany), 100 IU penicillin/mL and 100 μ g streptomycin/mL (Provitro®, Berlin, Germany).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Control