Journal: Cells

Article Title: Beyond Pattern Recognition: TLR2 Promotes Chemotaxis, Cell Adhesion, and Migration in THP-1 Cells

doi: 10.3390/cells12101425

Figure Lengend Snippet: Endothelial barrier disruption and transmigration in THP-1 WT and KO cells. ( a – h ) LECs were seeded onto ECIS arrays (96W20idf PET) to form an endothelial monolayer, and 1 × 10 5 THP-1 KO or WT cells were added ( a , b ), either without a ligand or with ( c , d ) Pam2, ( e , f ) Pam3, or ( g , h ) LPS. Monolayer disruption was documented in real time by impedance measurements using the ECIS system (9600Z). Time course diagram and bar graphs at 6 h after treatment showing mean values ± standard deviation (n = 4). A t -test was performed to assess differences between KO and WT cells (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: Endothelial cell monolayer disruption (transmigration) by monocytes +/− LPS and TLR2 ligand stimulation was measured using the 9600Z Electrical Cell-Substrate Impedance Sensing (ECIS) system (Applied BioPhysics, Troy, NY, USA).

Techniques: Transmigration Assay, Standard Deviation

Journal: medRxiv

Article Title: Lipidomics for diagnosis and prognosis of pulmonary hypertension

doi: 10.1101/2023.05.17.23289772

Figure Lengend Snippet: (A) Representative Bodipy fluorescence staining of hPASMC and hPAEC in the absence (Ctrl) or presence of FFA (scale bar: 50 µm). (B) Platelet-derived growth factor (PDGF)-BB induced proliferation of primary hPASMC measured with thymidine incorporation, in the absence (Ctrl) or presence of FFA (n = 4). Changes are expressed as percentages compared with untreated controls (Ctrl). (C) ACh-induced NO production in primary hPAEC (n = 4). Changes are expressed as percentages compared with untreated controls (Ctrl). (D) TEER, as determined by electrical cell-substrate impedance sensor (ECIS), shows a significant increase in hPAEC treated with FFA. Representative original curve (left panel) and changes expressed as percent change compared with controls (Ctrl) (n = 4). (E, F) Summarized data from hPASMC and hPAEC using the Seahorse XFe24 Extracellular Flux Analyzer. All measurements were performed on n = 25,000–50,000 cells/well and five wells per cell type. Each experimental group consisted of two to three patient cell lines (samples). All data were normalized to total protein per well before analysis. Results are given as median with interquartile range. Mann Whitney test * < 0.05, **< 0.01, ***< 0.001.

Article Snippet: TEER was determined using an electrical cell-substrate impedance sensor (ECIS) (Applied Biophysics, Troy, NY, USA).

Techniques: Fluorescence, Staining, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Circulating extracellular vesicles are associated with the clinical outcomes of sepsis

doi: 10.3389/fimmu.2023.1150564

Figure Lengend Snippet: Circulating EV-induced vascular permeability is associated with ARDS patients. Circulating total EVs were isolated from healthy controls (N=7) and septic patients (N=96). Among septic patients, 21 patients developed ARDS and 75 did not. These EVs at 1.5 x 10 10 EVs/ml were used in TEER assays which was measured in HMVEC for 6-24 hours by an ECIS instrument. Representative results from 7 healthy controls (A) and 7 septic patients (B, C) are shown. The association of TEER level with mortality (D) , ARDS development (E) and acute renal failure (F) were analyzed using Wilcoxon rank sum tests. Resistance levels are expressed as the level of impedance at 2 hours divided by the level of impedance prior to addition of EVs. Boxes present the 25 th , 50 th , and 75 th percentiles of resistance in each group. Whiskers extend 1.5 +/- the inner quartile range (i.e. difference between the 75 th and 25 th percentile). Grey points are the observed resistance values (% of original) for subjects within each group. EV, Extracellular vesicles; ARDS, acute respiratory distress syndrome; TEER, transendothelial electrical resistance; HMVEC, Human Lung Microvascular Endothelial Cell.

Article Snippet: Total circulating EVs at 1.5 x 10 10 /ml were added into each well and transendothelial electrical resistance (TEER) assays were performed using an ECIS instrument (Applied Biophysics).

Techniques: Permeability, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins

doi: 10.3390/ijms24076082

Figure Lengend Snippet: Schematic explaining ECIS theory. ( A ) Endothelial cells grown on the gold-plated electrodes of the ECIS plate that are coated with collagen, forming a confluent monolayer. The monolayer is formed via intercellular junctions and junctions with the collagen substrate, creating the paracellular and basolateral junctions, respectively. The formation of these junctions prevents high current flow from the electrode through the cells, where the red and green arrows represent the paracellular (Rb) and basolateral (alpha) current flow, respectively. This maintained electrical resistance is shown in graph C (Black). ( B ) The endothelial monolayer following treatment with factors that disrupt the endothelial barrier, such as melanoma cells or melanoma vesicular-bodies; the junctions weaken, allowing for greater current flow through the cells. This results in a decreased electrical resistance, shown in graph ( C ) (Blue). ( C ) A model ECIS graph depicting the plateau in resistance formed prior to treatment, indicating a fully formed endothelial barrier, and the corresponding results from 1. No treatment added (Black) and 2. Melanoma cell addition (Blue). Created with BioRender.com by Dayna Spurling; Reprinted/adapted with permission from Ref. . 2019, Anchan, A.

Article Snippet: To assess the strength of the cerebral endothelial barrier, hCMVECs were grown on gold-plated 96-well ECIS plates (Applied Biophysics, Troy, NY, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins

doi: 10.3390/ijms24076082

Figure Lengend Snippet: Human cerebral microvascular endothelial cell CD144 staining after NZM7-S treatment at varied concentrations. hCMVECs were seeded at 20,000 cells per well. NZM7-S were live stained with CytoTrack Red (CTR; red in image) and applied to the apical face of a confluent endothelial monolayer. Cells were co-cultured for 4 h, washed and then fixed. Junctions were stained with anti-CD144 (green). ( A ) An image from the media control well. ( B ) A paired ECIS experiment, where the boxed region shows the point of fixing for the ICC images (4 h). ( C , D,E ) The images of the melanoma-treated wells at different E:T ratios, noted on the left side of the panel. Images were acquired on the Operetta CLS imaging system. Full view images scale bar = 100 µm. Images are representative of 2 independent experiments.

Article Snippet: To assess the strength of the cerebral endothelial barrier, hCMVECs were grown on gold-plated 96-well ECIS plates (Applied Biophysics, Troy, NY, USA).

Techniques: Staining, Cell Culture, Imaging