Introduction
Angiogenesis is a physiological process in which new branches and the network of blood vessels are generated in the tissues. Although angiogenesis is essential for living bodies, it is also involved in a variety of diseases. For example, cancers induce the formation of vascular networks to supply energy and nutrient to themselves.
In vivo experiments using animal models have been prevalent for the research of angiogenesis. However, it is often difficult to obtain quantitative and reproducible data in such experiments, and the low throughout makes them unsuitable for drug discovery screening. On the other hand, in vitro culture assays are useful for drug screening because such systems allow strict control of the experimental conditions. CQ1 enables automated high-throughput in vitro assays. This application note shows an example of the analysis of the angiogenesis by cultured HUVEC in Matrigel.
Figure 1. Vascular network-like structure formed by HUVECs.
(A) A whole-well image showing the network formed by cultured HUVECs in Matrigel.
(B) Recognition by CQ1.
(C and D) Higher magnification images of the areas (white frames) in (A) and (B), respectively.
Experimental procedures
Human umbilical vein endothelial cells (HUVECs) were incubated in the serum starvation condition (1 % FBS) for 24 hours.
Cells were seeded in a Matrigel-coated, 96-well microplate (10,000 cells/well).
After incubation (4 hours) to allow them to form blood vessel-like networks, Suramin (0 – 50 µM) and CellTracker Red (5 µM) were added to the culture medium and cells were incubated for an hour at 37℃.
Images were acquired with CQ1 (4 x objective, 561 nm laser). Maximum intensity projection (MIP) images were constructed from 12 z slices (764 µm from bottom to top), then whole-well images were reconstructed from four adjacent images. The images were analyzed in the CQ1 software.
Figure 2. Effects of Suramin
Suramin is a reagent which is known as an inhibitor of angiogenesis in a dose-dependent manner. (A) Control well. The network formed by HUVECs covers the whole-well. (B) The well treated with 50 µM Suramin. The network is largely disrupted.
Figure 3. Dose-response curve for the effects of Suramin
Images were analyzed using Skelton function in the CQ1 software and dose-response curves were constructed for the effects of Suramin on length of the line objects (left), the number of branching points (middle) and number of branches (right) per well.
Results and discussion
The effects of Suramin on the vascular network-like structure formed by cultured HUVECs were analyzed and quantified using CQ1. Users can easily make automated procedures from image acquisition through image analysis in CQ1. By using such procedures, users can effectively collect quantitative and reproducible data in high throughput.
Even in the case of the samples like the vascular network in Matrigel, where cells are distributed in the coating material with uneven thickness, CQ1 can capture the complete three-dimensional structure of the network by acquiring z-stack images without missing out-of-focus cells.
The microlens-enhanced dual Nipkow disk confocal system in CQ1 cause only a very low level of photobleaching and phototoxicity on the samples. This feature allows repeated image acquisitions in CQ1 with a minimal damage to the samples.
A tiled image which consists of four fields acquired with the 4 x objective lens in CQ1 covers a whole-well in a 96-well microplate. CQ1 software can generate a large field image by stitching images of adjacent fields. Analysis of the tiled images would provide more realistic results avoiding possible errors caused by analyzing each field separately.
The dynamic process of angiogenesis can be investigated in the time-lapse live cell imaging using CQ1 with optional environmental control system.
Our Social Medias
We post our information to the following SNSs. Please follow us.
| Follow us | Share our application | |
| @Yokogawa_LS | Share on Twitter | |
| Yokogawa Life Science | Share on Facebook | |
| Yokogawa Life Science | Share on LinkedIn |
Yokogawa's Official Social Media Account List
相关产品&解决方案
-
CellPathfinder
CellPathfinder is designed for our HCA systems, CQ1 and the CellVoyager series. From beginners to experts, the analysis software lets you quantify subtle physiological changes and even label-free samples with various graph options.
-
CellVoyager High-Content Analysis System CQ3000
CQ3000 can acquire high-resolution 3D images at high speed while culturing cells by combining options according to the application.
-
CQ1台式高内涵分析系统
CellVoyager CQ1采用节省空间的台式设计，提供高质量的共聚焦图像和延长的活细胞成像。
-
CV8000高内涵筛选系统
CellVoyager CV8000是先进的高内涵筛选系统。改进的内置培养箱可以让客户分析长期的活细胞反应。凭借其可扩展性、4个摄像头、5个激光器和可选的内置移液器，该系统允许日益复杂的分析开发和高内涵筛选。
-
生命科学
解锁生命本质
横河电机灵敏、高精度的解决方案可实现快速准确的测量，支持突破性的研究，加快药物发现，并优化大规模生物生产。
-
高内涵分析
我们的高内涵分析系统利用强大的分析软件助力从基础科学到药物发现筛选的一系列研究应用。
