Tube Formation Assay for Angiogenesis

Tube formation assay was performed as previously reported^{46 (link)}. Briefly, a total of 35 × 10⁴ HUVECs were seeded in Matrigel-coated (Corning, USA) 96-well plates in DMEM-F12 including 1% EV-free FBS. The cells were treated with either 0.5 µg EVs derived from control or ticagrelor treated H9c2 cells. The tube formation was visualized at 6 h and 24 h of incubation by an inverted light microscope (Zeiss, Germany). Branche, mesh, segment, and node formations were analyzed by Angiogenesis Analyzer plug-in for ImageJ (NIH, USA)^{47 (link)}. Changes in angiogenesis on EV-treated cells were expressed as a percentage of the controls (untreated cells). Values represent the mean (± SD) of triplicate formation assay.

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Bitirim C.V., Ozer Z.B., Aydos D., Genc K., Demirsoy S., Akcali K.C, & Turan B. (2022). Cardioprotective effect of extracellular vesicles derived from ticagrelor-pretreated cardiomyocyte on hyperglycemic cardiomyocytes through alleviation of oxidative and endoplasmic reticulum stress. Scientific Reports, 12, 5651.

Publication 2022

Matrigel-coated inverted light microscope

Angiogenesis Assay Cells Light microscope Matrigel Ticagrelor

Corresponding Organization :

Other organizations : Ankara University, Pennsylvania State University

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Variable analysis

independent variables

EV treatment: Control vs. Ticagrelor-treated H9c2 cells

dependent variables

Tube formation (Branches, mesh, segments, and nodes)

control variables

Cell type: HUVECs
Cell seeding density: 35 × 10^4 HUVECs
Culture substrate: Matrigel-coated 96-well plates
Culture medium: DMEM-F12 with 1% EV-free FBS
Incubation time: 6 h and 24 h

controls

Positive control: Not explicitly mentioned
Negative control: Untreated HUVECs

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