HPAECs (Lonza) were cultured in EBM-2 BulletKit media. Active Hla was prepared as described (Wilke and Bubeck Wardenburg, 2010 (link)). HPAECs exposed to Hla or HlaH35L (5 μg/ml) were stained with α-vWF antibody or exposed to platelets under shear stress of 150 s−1 after plating in IBIDI μ-slide VI flow chambers for platelet binding quantification. Cells were examined on an Olympus DSU Spinning Disk Confocal Microscope.
μ slide 6
Manufactured by Ibidi
Sourced in Germany
The μ-Slide VI is a specialized lab equipment designed for cell culture applications. It provides a six-well format for culturing cells, allowing for multiple experiments to be conducted simultaneously. The device features a unique structure and material composition to facilitate optimal cell growth and observation.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert fluorescent microscope, by Zeiss (6 mentions)
Orca er digital camera, by Hamamatsu Photonics (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Dsu spinning disk confocal microscope, by Olympus (1 mentions)
Hpaec, by Lonza (1 mentions)
Celltracker green, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Fv1000 confocal laser microscope, by Olympus (1 mentions)
Phalloidin tritc, by Thermo Fisher Scientific (1 mentions)
Hal 100 microscope, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Laser confocal microscope, by Leica Microsystems (1 mentions)
Ab2893, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Rabbit anti β1 integrin, by Santa Cruz Biotechnology (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Mouse anti talin, by Merck Group (1 mentions)
Eight well μ slide, by Ibidi (1 mentions)
Mouse anti cortactin, by Merck Group (1 mentions)
10 protocols using μ slide 6
1
Quantification of Hla-induced Platelet Binding
2
Co-culture of Endothelial and Mesenchymal Stem Cells
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Primary EC were dissociated using trypsin/EDTA (Sigma) and seeded in pre-coated channel slides (μ-Slide VI; ibidi GmbH, Martinsried, Germany) at a density that would yield a confluent monolayer within 24h [4 (link), 33 (link)]. After 24h, MSC were dissociated, counted and labelled with 5μM Cell Tracker Green (Life Technologies) for 30min (1.5x105 cells/ml in MSCGM), and seeded onto the EC (~13,500 MSC per channel) and allowed to settle for 1h as described [4 (link), 33 (link)]. Non-adherent cells were removed by washing with MSCGM, and the numbers of EC and of fluorescent MSC were counted in 5 fields (Fig 2A ). The ratio of MSC:EC was then calculated. Cells were co-cultured in direct contact for 24h prior to treatment with or without 100U/ml tumour necrosis factor–alpha (TNFα; R&D Systems, Abingdon, UK) for a further 4h. EC mono-cultures were set up in parallel as controls.
3
Fura-2 calcium imaging of VEGF-treated cells
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Cells were incubated with fura-2AM for 1 h at 37 °C followed by a 0.5 h wash at room temperature (21± 2 °C) on Ibidi μ-Slide VI 0.4. Measurements were made at room temperature on a Zeiss Axiovert fluorescent microscope equipped with x20 (NA 0.75) or x40 (NA 1.3, oil) Fluar objective and excitation light from a xenon lamp selected by a monochromator (Till photonics, Germany). Emitted light was collected via an emission filter and images captured by an Orca-ER digital camera (Hamamatsu, Japan). The extracellular (superfusion) recording solution contained (mM): 130 NaCl, 5 KCl, 8 D-glucose, 10 Hepes, 1.2 MgCl2, 1.5 CaCl2, titrated to pH 7.4 with NaOH. For the experiment of Extended Data Fig 4a-b , 2 ng.mL−1 VEGF was included. The change (Δ) in intracellular calcium (Ca2+) concentration above baseline is shown by the ratio of fura-2 fluorescence (F) emission intensities for 340 and 380 nm excitation (ΔF ratio). For statistical comparisons, the total ΔF ratio above baseline was calculated for each shear stress and divided by the time period over which the ΔF ratio was measured (ΔF ratio.s−1).
4
Fura-2 calcium imaging of VEGF-treated cells
5
Immunofluorescence Staining of Cell Adhesion Proteins
6
X-Irradiation-Induced DNA Damage Assay
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Cells were exposed to 6 Gy X-IR and allowed to recover after 48 h of incubation. Cells grown in a μ-slide VI (Ibidi, Martinsreid, Germany) were fixed using 4% paraformaldehyde for 30 min at 4°C and permeabilized with PBS containing 0.2% Triton X-100 for 10 min. Cells were then blocked with 3% BSA and incubated with the rabbit polyclonal antibody to γH2AX (ab2893, 1:100, Abcam, Cambridge, MA, USA). Following washing, the cells were incubated with the Alexa Fluor 488 goat anti-rabbit IgG (#4412S, 1:2,000, Cell Signaling Technology, Danvers, MA, USA) or 1 h at 37°C. The nuclei of cells were subsequently stained with DAPI. Immunofluorescence images were obtained using a laser confocal microscope (Leica Microsystems, Wetzlar, Germany).
7
Neutrophil Chemotaxis Assay Protocol
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Via time-lapse microscopy, the μ-slide chemotaxis assay allows to study directionality, velocity, and total distance covered during the migration of PMNs toward a concentration gradient of chemokines (13 (link)). A microfluidic chamber (μ-slide VI, IBIDI, München, Germany) was used to create a stable concentration gradient of CXCL8. PMNs of patients with PCD and adult controls (3 × 106 cells/ml) were suspended in RPMI 1640 + 2 mM HEPES + 0.5% HSA (μ-slide chemotaxis buffer) and after injection of the cells in the channel, the microfluidic chamber was incubated at 37°C for 30 min to allow the PMNs to settle down. Perpendicular on the channel with the cells, a concentration gradient of CXCL8 (200 ng/ml in μ-slide chemotaxis buffer) was created. Every 90 s, a snapshot of the cells was made with an inverted microscope (10× phase-contrast objective; Zeiss Axiovert 200 M) for 2 h. Constant temperature (37°C) and CO2 concentration (5%) were maintained throughout the recording. Migration of 20 randomly picked PMNs of each donor was tracked with the ImageJ manual tracking plug-in and data were analyzed with the IBIDI chemotaxis and migration tool. The optimal concentration of CXCL8 was determined in pilot experiments with healthy neutrophils.
8
Imaging Immune Cell-Endothelial Interactions
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siCD73‐silenced and control HDLECs were seeded onto IBIDI chamber slides (μ‐Slide VI, IBIDI, Martinsried, Germany). One day later, either immature or moDCs matured for 6 h with LPS/IFN‐γ were labeled with CFSE (Thermo Fisher Scientific) and added to the slides (5000 cells/lane). After 45 min, nonadherent cells were rinsed away and the remaining cells fixed with paraformaldehyde. Slides were recorded with a total internal reflection microscopy microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and analyzed with ImageJ (Fiji, NIH, Bethesda, USA).
9
ECM-Mediated Adhesion of LO-EPCs
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100 μg/μL of collagen IV, collagen I, fibronectin, vitronectin, or laminin was preplated to Ibidi μ-Slide VI 0.4 Luer slides for 1 hour at 37°C before rinsing twice with DPBS. 2 × 104 LO-EPC were seeded on each ECM-treated Ibidi slide and incubated at 37°C for 45 min. After washing twice with DPBS, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and stained with Hoechst for 30 min. The total number of adherent cells was imaged, counted, and expressed as adherent cells per square millimetre.
10
Cell Migration Assay Reagents
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Rabbit anti–β1 integrin, goat anti-CAPN1, and rabbit anti-FAK were from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-ezrin was purchased from Cell Signaling (Beverly, MA). Rabbit anti–phospho-Y397 FAK and mouse anti-paxillin were from BD Transduction Labs (Mississauga, Canada). Mouse anti-cortactin was purchased from EMD Millipore (Etobicoke, Canada). Mouse anti–γ-tubulin, mouse anti-moesin, mouse anti-vinculin, mouse anti-talin, and rabbit anti-zyxin were purchased from Sigma-Aldrich (Oakville, Canada). Rabbit anti–calpain-2, which recognizes both CAPN2 and CAPNS1, was provided by P. Greer (Queen’s University, Kingston, Canada; Arthur et al., 2000 (link)). Transwell chambers (8-μm pore size) were obtained from Corning (Mississauga, Canada). Eight-well chamber slides were purchased from LabTek (Mississauga, Canada). μ-Slide VI, 35-mm μ-Dish, and eight-well μ-Slides were from Ibidi (Madison, WI). Alexa Fluor reagents and cell trackers were obtained from Life Technologies (Burlington, Canada). Growth factor–reduced Matrigel and rat-tail collagen I were purchased from BD Biosciences (Mississauga, Canada). Fibronectin, gelatin, and puromycin were from Sigma-Aldrich.
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