Journal: Journal of Neuroinflammation

Article Title: Unique and shared inflammatory profiles of human brain endothelia and pericytes

doi: 10.1186/s12974-018-1167-8

Figure Lengend Snippet: Generation of pure endothelial cultures from human brain tissue. Brain tissue was cultured to generate mixed glial and endothelial cultures. Cells were fixed, and purity of cultures was assessed at passage two using immunocytochemistry. a Processing of biopsy specimens, followed by b mechanical and c – e enzymatic digestion at 37 °C. Suspension was f triturated and g passed through cell strainers h which were washed. i Debris were collected, spun, and j plated onto a matrigel-coated flask. k Schematic of the method used to generate mixed glial (containing astrocytes/microglia/endothelia/pericytes), and isolated endothelial cultures. l Representative live images of passage 1 cultures throughout different growth stages. Arrowheads highlight blood vessels and endothelia while areas outlined in red are endothelial colonies, arrows indicate astrocytes and asterisks denote microglia. Scale bar = 200 μm. m Representative images and n quantification of staining for lineage markers (CD31, endothelial; PDGFRβ, pericyte; GFAP, astrocyte; PU.1, microglial) in passage two mixed glial and endothelial cultures. Scale bar = 500 μm, inset scale bar = 100 μm. d Cell lineage assessment of endothelial and mixed glial cultures (mean, n = 3 mixed glial, n = 5 endothelial)

Article Snippet: At the first passage, Accutase (Gibco, CA, USA) was used to dissociate cultures, before being washed in MACS buffer (1% bovine serum albumin, 1 mM EDTA in PBS) and Fc receptor blocking agent added, followed by addition of magnetic bead-conjugated mouse IgG1 anti-human CD31 antibody (Miltenyl Biotech, Cologne, Germany) for 15 min at 4 °C.

Techniques: Cell Culture, Immunocytochemistry, Suspension, Isolation, Staining

Journal: Journal of Neuroinflammation

Article Title: Unique and shared inflammatory profiles of human brain endothelia and pericytes

doi: 10.1186/s12974-018-1167-8

Figure Lengend Snippet: Isolated endothelia are proliferative and re-establish blood-brain barrier phenotype. Endothelial and pure pericyte cultures were treated with EdU (48 h, 10 μM) then fixed and stained for the junctional proteins ZO-1, claudin-5, CD31, endothelial nuclear marker ERG, and proliferation marker EdU. RNA was extracted from endothelia, and a single pure pericyte culture, and gene expression analysed by qPCR. a Representative images of immunocytochemical staining of zona occludens-1 (ZO-1), CD31, claudin-5, and ERG in endothelia and pericytes. Scale bar = 500 um. b High-magnification representative images of co-staining of CD31 and claudin-5, and c CD31 and claudin-5. Scale bar = 25 μm. d Gene expression analysis of endothelial cultures (mean ± SEM, n = 3), normalised to gene expression of a single pure pericyte culture. e Representative images of EdU staining in endothelia and f quantification of basal proliferation over 48 h

Article Snippet: At the first passage, Accutase (Gibco, CA, USA) was used to dissociate cultures, before being washed in MACS buffer (1% bovine serum albumin, 1 mM EDTA in PBS) and Fc receptor blocking agent added, followed by addition of magnetic bead-conjugated mouse IgG1 anti-human CD31 antibody (Miltenyl Biotech, Cologne, Germany) for 15 min at 4 °C.

Techniques: Isolation, Staining, Marker, Gene Expression

Journal: Journal of biotechnology

Article Title: Expediting adenovirus titer assays via an algorithmic live-cell imaging technique.

doi: 10.1016/j.jbiotec.2024.09.018

Figure Lengend Snippet: Figure 1. Timelines for the proposed Incucyte assay and the traditional Hexon staining assay. Plates seeded with an HEK 293 cell monolayer were infected with a dilution series created from Ad-GFP virus samples. After a 48-hour incubation period, the cell monolayer was imaged via a Sartorius Incucyte live-cell imaging system, and the FRs corresponding to infected cells were counted using the Incucyte’s segmentation algorithms. The same plates were subjected to the Hexon staining assay, imaged, and the stained regions were counted by the operator.

Article Snippet: [27] I. Essen Bioscience, “IncuCyte Live-Cell Analysis Systems User Manual.” Sartorius, Inc, 2022.

Techniques: Staining, Infection, Virus, Incubation, Live Cell Imaging

Journal: Journal of biotechnology

Article Title: Expediting adenovirus titer assays via an algorithmic live-cell imaging technique.

doi: 10.1016/j.jbiotec.2024.09.018

Figure Lengend Snippet: Figure 4. FR counts for the same image under different edge sensitivities and minimum mean intensities. FRs identified by the Incucyte segmentation algorithm are outlined in pink. The total number of identified FRs in each image is shown in the attached black box. The yellow and cyan squares are used to outline specific FRs across all parameter conditions. The above image was acquired using the “Autoscale Well” setting and the 24-well plate used in R3. It depicts a cellular monolayer infected with a viral solution dilution factor of 7 (see Supplemental Information, Section 2, for more information regarding the imaging layout).

Article Snippet: [27] I. Essen Bioscience, “IncuCyte Live-Cell Analysis Systems User Manual.” Sartorius, Inc, 2022.

Techniques: Infection, Imaging

Journal: Journal of biotechnology

Article Title: Expediting adenovirus titer assays via an algorithmic live-cell imaging technique.

doi: 10.1016/j.jbiotec.2024.09.018

Figure Lengend Snippet: Figure 7. Comparison of infectious titer estimates attained via Incucyte and Hexon staining for various samples (R1 to R6).

Article Snippet: [27] I. Essen Bioscience, “IncuCyte Live-Cell Analysis Systems User Manual.” Sartorius, Inc, 2022.

Techniques: Comparison, Staining

Journal: Journal of biotechnology

Article Title: Expediting adenovirus titer assays via an algorithmic live-cell imaging technique.

doi: 10.1016/j.jbiotec.2024.09.018

Figure Lengend Snippet: Figure 8. FR counts obtained via Incucyte analysis for the dilution series plated on 24-well and 96-well plates for R5. The 96-well data includes samples from dilution factors 4 to 9, while the 24-well data includes samples from dilution factors 6 to 9, enabling a comparison of half-dilution factors from the two datasets. The FR count in the 96-well plate plateaus from dilution factors 4-6, and the normalized standard deviation increases as the count number falls below 200. The count range for the 24-well plate, identified as 300-6500 in prior 24-well runs, is shown in red at the bottom right graph. Both plates were infected in accordance with the plate map depicted in Figure S1 of the Supplemental Information.

Article Snippet: [27] I. Essen Bioscience, “IncuCyte Live-Cell Analysis Systems User Manual.” Sartorius, Inc, 2022.

Techniques: Comparison, Standard Deviation, Infection

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A ) Overlay histograms of LFA-1 diffusion on monocytes (4578 trajectories), resting mDCs (26756 trajectories) and CCL21 activated mDCs (4213 trajectories). ( B ) Percentage of total mobile LFA-1 population (normalized to 100%) displaying slow and fast diffusion on monocytes, mDCs and 2 min CCL21 activated mDCs. ( C ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on monocytes, mDCs and 2 min CCL21 activated mDCs. ( D ) Stationary fraction of LFA-1 on monocytes, mDCs and CCL21 activated mDCs, displayed as the difference from the total stationary fraction on mDCs, which serve here as the default. Data from A–D on CCL21 activated mDCs is based on 22 cells in independent experiments from 3 different donors. ( E ) Percentage of the stationary, slow and fast diffusing LFA-1 molecules at different time points after CCL21 activation. ( F ) D values for the total mobile, and slow and fast fractions of LFA-1 at different time points after CCL21 activation. Data from E and F is based on 11 cells, 11 independent samples and around 2000 trajectories per time point. A – F Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns (P >0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Activation Assay, Comparison

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A,B ) ICAM-1 (either monomeric: ICAMm, or as nano-aggregates: ICAMagg) was added to resting mDCs and mobility was measured before, and between 1 and 5 min after addition. ( A ) Stationary fraction of LFA-1 molecules on resting mDCs and mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs, which serve here as the default. ( B ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs and after addition of either monomeric ICAM-1 or ICAM-1 nano-aggregates. 30 cells divided over 2 independent experiments (3393 trajectories) were imaged for the ICAMm condition and 10 cells (684 trajectories) for ICAMagg. ( C, D ) ICAM-1 (either monomeric or nano-aggregates) was added together with CCL21 to mDCs and mobility was measured before, and 2 minutes after addition. ( C ) Stationary fraction of LFA-1 molecules on resting mDCs (serving as reference control), CCL21 activated mDCs and CCL21 activated mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs. ( D ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs, CCL21 activated mDCs and after simultaneous addition of CCL21 and either monomeric ICAM-1 or ICAM-1 nano-aggregates. 16 cells (8423 trajectories) from 2 different donors divided over 5 independent samples (ICAMm) and 7 cells (314 trajectories) from 2 different donors divided over 7 independent samples (ICAMagg) were imaged. ( E ) Quantification of fluorescent ICAM-1 dimers (monomers bound together due to antibody labelling) and nano-aggregates binding in resting and CCL21 activated mDCs, normalized to the area quantified and to the background signal outside of the cell. For this, regions of the cell in between the obvious fluorescent ICAM-1 aggregates were selected, the fluorescent intensity was measured using ImageJ, and used to compare the baseline fluorescent signal across all 4 conditions. 20 cells per condition were imaged. ( A–E ) Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05), ** (P<0.001) and *** (P<0.0001). ( F ) Quantification of bound ICAM-1 nano-aggregates to resting and CCL21 activated mDCs. After applying a threshold of 25% of the fluorescent signal, all visible fluorescent spots per cell were counted. 20 cells per donor and 3 different donors were imaged. Each data point represents the mean value for 1 donor. Means ± SEM and individual data points are depicted, and dotted lines connecting datapoint of the same experiment indicate that not just in average, but in each individual experiment using a different donor, an increase of ICAM-1 nano-aggregate binding is observed after CCL21 activation. The paired two-tailed Student T-test was used to determine significant differences between means. ( G–I ) Representative examples of confocal images of ICAM-1 binding to mDCs: ( G ) dimeric ICAM-1 to resting cells, ( H ) nano-aggregates of ICAM-1 to resting cells and ( I ) nano-aggregates to CCL21 activated cells. Arrows in H and I point to the binding of individual ICAM-1 nano-aggregates to LFA-1.

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Control, Binding Assay, Comparison, Activation Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A, B ) Representative images of ( A ) a monocyte seeded on a TS2/4 pattern, ( B ) a resting mDC on irrelevant IgG1 pattern, ( C ) a resting mDC seeded on a TS2/4 pattern and ( D ) a CCL21 activated mDC seeded on a ICAM-1 pattern. Green corresponds to Talin1 and red to the location of IgG1, TS2/4 or ICAM-1 positive squares. Cells are delineated by white lines. ( E ) Quantification of the degree of Talin1 enhancement to the positive areas in monocytes in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( F ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in monocytes. An enhancement factor of ≥1.5 was considered significantly enhanced, since 95% of the control sample (monocytes on IgG1) showed an enhancement factor below this value. ( G ) Quantification of the degree of Talin1 enhancement to the positive areas in mDCs in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( H ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in mDCs. Around 60 cells of 3 different donors were analyzed per condition. Monocytes contained 10 positive areas on average per cell, while mDCs contained around 50 positive areas. Means ± SEM are depicted. The Kruskal-Wallis test, followed by Dunn’s multiple comparison test was used to determine significant differences between means in E and G. The One-way ANOVA followed by the Tukey’s multiple comparison test were used to determine significant differences between means in F and H. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Control, Comparison