1.2 Matrigel Plug
Assay
The Matrigel plug assay is a widely used in vivo assay, which allows
the evaluation of angiogenesis within a few days. Matrigel is a
soluble basement membrane extract of the Engelbreth-Holm-
Swarm mouse sarcoma, which includes the components laminin,
collagen IV, heparan sulfate proteoglycan, and entactin. It is liquid
at 4C, reconstitutes into a gel when injected subcutaneously into
mice, and finally forms a solid plug, into which endothelial cells can
immigrate and form vessels. The angiogenic process can be modu-
lated by adding growth factors or pro- and antiangiogenic drugs to
the Matrigel [12–14]. The angiogenic response is quantified after
harvesting and sectioning the plugs by assessing CD31-positive
structures in immunofluorescent stainings. Alternatively, cells can
be released from the plugs by mild protease treatment and analyzed
by flow cytometry [15]. Matrigel has the advantage that angiogen-
esis is initiated in a natural environment. However, since it contains
endogenous growth factors and cytokines itself (even if the growth
factor-reduced Matrigel is used), interference with test compounds
and spontaneous sprouting can occur. We employed the Matrigel
plug assay to investigate angiogenesis in AMPKα1 knockout
mice [2].2 Materials
2.1 Endothelial Cell
Spheroid Assay
2.1.1 siRNA-Mediated
Downregulation of AMPKα
in HUVEC
- HUVEC: Primary HUVEC isolated from anonymously
acquired umbilical cords according to the Declaration of Hel-
sinki ‘Ethical Principles for Medical Research Involving
Human Subjects’ (1964) (seeNote 1). - Endothelial cell growth medium: Medium 199, 17.5% (v/v)
fetal calf serum (FCS), 2.5% (v/v) human serum, 7.5μg/mL
endothelial mitogen, 7.5 U/mL heparin, 680μM glutamine,
100 U/mL penicillin, 100μg/mL streptomycin, 100μM
ascorbic acid (seeNote 2). - Hank’s balanced salt solution (HBSS).
- M199/HSA: Medium 199 with 0.25% (w/v) human serum
albumin. - siRNA duplex oligonucleotides based on human cDNA encod-
ing AMPKα1 or AMPKα2(seeNote 3). - Transfection kit: SAINT-sRNA transfection reagent (seeNote
4 ). - HEPES-buffered saline (HBS): 20 mM HEPES, 150 mM
NaCl, pH 7.4. - Cell culture incubator (5% CO 2 ,37C, 100% humidity).
2.1.2 Generation of
Spheroids
- Methylcellulose stock solution: 1.2% (w/v) methylcellulose.
Weigh 6 g of methylcellulose, transfer to a 500 mL glass bottle,
Angiogenesis 521