The tubulogenesis assay was performed as described in Arnaoutova and Kleinman (2010 (link)), with slight alterations. Primary human myocardial microvascular endothelial cells (HMVEC-Cs), a potential clinical target of angiogenic mechanisms after MI, were maintained in EGM2-MV media (both from Lonza) and used at Passage 6. Matrigel growth factor reduced (10 μL, Corning) was used to coat a 15-well Angiogenesis μ-Slide (81506; Ibidi) and allowed to polymerize at 37°C for 30 min. HMVEC-C were suspended in complete media (EGM2-MV), Conditioned Media (CM) or concentrated negative control (a-MEM no cells) diluted in basal media (EBM), with a final CM concentration of 5x, and seeded at 6.5 x 104 cells/cm2 in a total of 50 μL per well. Conditioned media from 3 independent hypoxia inductions was run in parallel, along with technical triplicate wells for each condition. After 7.5 h incubation at 37°C and 5%CO2 the center of each well was imaged using phase contrast microscopy on an inverted microscope Axiovert 200 (Carl Zeiss) with a 10x objective. Image analysis was performed on ImageJ (NIH) using the Angiogenesis Analyzer plugin (Carpentier, 2012 ).
μ slide angiogenesis
Manufactured by Ibidi
Sourced in Germany, United States, United Kingdom
The μ-Slide Angiogenesis is a laboratory equipment designed for the study of angiogenesis, the process of new blood vessel formation. It provides a controlled environment for the observation and analysis of this biological phenomenon.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (20 mentions)
Matrigel, by Corning (6 mentions)
Growth factor reduced matrigel, by Corning (3 mentions)
Growth factor reduced matrigel, by BD (3 mentions)
Calcein am, by Merck Group (3 mentions)
Matrigel, by Ibidi (3 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (2 mentions)
Vegf165, by R&D Systems (2 mentions)
Vectorshield mounting media with dapi, by Vector Laboratories (2 mentions)
Ulex europaeus agglutinin 1, by Vector Laboratories (2 mentions)
Ebm 2, by Lonza (2 mentions)
Axiovert 200, by Zeiss (2 mentions)
Matrigel growth factor reduced basement membrane matrix, by Ibidi (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Phase contrast microscope, by Nikon (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Tcs sp8 confocal microscope, by Leica camera (2 mentions)
Huvecs, by Ibidi (2 mentions)
Cell culture media, by Thermo Fisher Scientific (2 mentions)
Axiovision program, by Zeiss (2 mentions)
73 protocols using μ slide angiogenesis
1
Tubulogenesis Assay for Endothelial Cell Angiogenesis
2
Angiogenic Potential of SEC
3
RPE-Derived EV Modulation of ECFC Angiogenesis
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Endothelial colony forming cells (ECFCs) cultured as a cell monolayer were treated overnight with apical and basal low‐risk and high‐risk RPE EVs. EVs were derived from cell conditioned media from 200,000 RPE cells (per replicate) to model 1:1 ratio of exposure RPE:ECFCs. Additionally, to assess the functional effect of EVs, dose‐response experiments were carried out at different ratios of RPE EVs – ECFCs (i.e., 1:4 and 1:2). Exosome depleted media was used to harvest EVs secreted by RPE cells over 72 h and EVs were purified using size exclusion chromatography as described in Section 2.2 . After treatment, ECFCs were detached and counted. The angiogenesis μ‐Slide (Ibidi) was used to assess 3D tube formation capacity. An ECFC suspension was mixed in a 2:3 ratio with Matrigel (Corning) and dispensed into slide to form a 10 μl Matrigel droplet containing 15,000 cells (final density of 1.5 million ECFCs per ml). angiogenesis μ‐Slides were left in the incubator for 30 min to enable Matrigel polymerisation, and then covered with 50 μl of EGM‐2 media (Lonza). Tube‐like structures were assessed after 48 h, using the EVOS Cell Imaging system (Thermo Fisher). Analysis of the tube area was performed using ImageJ.
4
Angiogenesis Assay with LATS1/2 Knockdown
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M010817 cells were pretreated with siRNA targeting siLATS1/2 or non-targeting control and TGFβ or Wnt-3a for 5 d as described above. 3.4 × 104 cells in 50 μl medium containing TGFβ or Wnt-3a where appropriate were seeded into angiogenesis μ-Slide (Ibidi) coated with 10 μl growth factor-reduced Matrigel (diluted 1 + 2; BD Biosciences, 356230). Cells were incubated for 4 h and imaged using a Leica DMIL phase-contrast microscope with 10×, 0.22 NA objective. Images were processed with ImageJ (ImageJ, Wayne Rasband, National Institutes of Health).
5
3D Angiogenesis Assay with BMP9 and VEGF
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PAECs were cultured in a collagen and fibronectin gel matrix as previously described50 (link). Briefly, cells were combined with the gel at a final concentration of 1×106. The suspension was then pipetted into an angiogenesis μ-slide (Ibidi, Germany) in a volume of 10 μl/well. After 10 minutes at 37°C to allow polymerization, the gel was overlaid with 40μl of media (EBM-2, Lonza and 2% FBS), with or without 5 ng/ml BMP9 or 30 ng/mL VEGF165 (both R&D Systems) individually or combined. The 3-D cultures were left at 37°C for 24 hours and tube formation confirmed by brightfield microscopy. Images were collected and parameters measured and quantified using Image J software. In order to gain confocal images, gels were first fixed in 4% PFA, washed in PBS and incubated with Rhodamine labelled ULEX Europaeus Agglutinin1 (Vector Laboratories, Burlingame, CA) overnight at 4°C. After 3× 10 minute PBS washes, nuclear staining was carried out using 10μl of VectorSHIELD mounting media with DAPI (Vector Laboratories) for 30 minutes. A final x3 wash in PBS was completed.
6
Phenotypic and Functional Analysis of Bone Marrow-Derived Macrophages
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Single-donor BMCs (107) or pooled BMCs (75×106) of three individual donors were sorted (FACSAria III; BD) after staining for CD14/CD16/CD45 and plated on 24-well plates or 4-well chamber slides in RPMI 1640 medium with 10% FCS and antibiotics. Phagocytosis of FITC-conjugated latex beads (1∶1000; Sigma-Aldrich) and hydroethidine (10 µg/ml; Invitrogen, Darmstadt, Germany) staining for spontaneous intracellular superoxide production in PBMC- or sorted BMC-derived macrophages were determined by flow cytometry after 24 hours in culture. Cytoskeletal filaments in cultured BMCs were visualized by fluorescence microscopy (Leica DM6000B, Wetzlar, Germany) after cell fixation/permeabilization (Cytofix/Cytoperm; BD) following incubation with anti-α-tubulin-FITC (Sigma-Aldrich) mAb and rhodamine-phalloidin (Biotium, Hayward, CA). Vectashield with DAPI (Vector Laboratories, Peterborough, UK) was used as mounting medium and to stain cell nuclei. In another experimental setup the sorted BMCs were cultured in angiogenesis μ-slide (IBIDI, Martinsried, Germany) on Matrigel (BD) supplemented with 50 ng/ml VEGF and 100 ng/ml Cxcl12 (both from Peprotech, Hamburg, Germany).
7
Tubule Formation Assay with HMEC-1
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Tubule forming capacity was measured using HMEC-1 cells (passage 16). CM was layered on top of 10 μl solidified growth factor reduced Matrigel (BD, Vianen, NL) on an IBIDI angiogenesis μ-Slide (München, DE). 10,000 HMEC-1 cells were resuspended in serum-free α-MEM and seeded into the CM. The assay was performed in triplicate. After 16 hours, microscopic pictures were taken at 4x magnification. Tubule networks were manually traced in Adobe Photoshop and subsequently analyzed using the AngioAnalyzer ImageJ plugin, which provided the junction-to-tubule length ratio as a measure for network maturity. Values are presented as relative to the positive control and normalized per experiment.
8
3D Angiogenesis Assay with BMP9 and VEGF
9
Angiogenesis Assay using HUVECs
10
3D Angiogenesis Tube Formation Assay
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The angiogenesis μ-Slide (Ibidi) was used to assess 3D tube formation capacity. An HRMEC suspension was mixed in a 2:3 ratio with Matrigel (Corning) and dispensed into slide to form a 10 μl Matrigel droplet containing 20,000 cells (final density of 2 million HRMECs/ml). angiogenesis μ-Slides were left in the incubator for 30 min to enable Matrigel polymerization, and then covered with 50 μl of ECM media (Innoprot). ECM was supplemented to a final concentration of 25 mM D-glucose for high glucose conditions. ECM supplemented with 20 mM l -Glucose was used as the osmotic control. Tube-like structures were assessed after 24 h, using the EVOS Cell Imaging system (Thermo Fisher). Analysis of the tube area was performed using Fiji‐ImageJ2 software (Schindelin et al., 2012 (link)).
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