3.2 Matrigel Plug
Assay
3.2.1 Mouse Model
- Perform experiments with sex-matched 3-month-old wild-type
and AMPKα1 knockout mice. Carry out all animal procedures
in accordance with the guidelines of the local and national
committees for animal experiments. - Use a mouse identification system (i.e., ear punch or tag,
tattoo).
3.2.2 Generation
of Matrigel Plugs in Mice
- Calculate the amount of Matrigel needed for your experiment.
For adult mice 2 500 μL per mouse are required to generate
control and experimentally treated plugs, respectively (seeNote
44 ). Thaw the Matrigel overnight in an ice bath.
Fig. 2VEGF induces sprouting in HUVEC spheroids. Embedded HUVEC spheroids were treated with 50 nM
sunitinib, a VEGF receptor 2 inhibitor, or vehicle. After 30 min, 10 ng/mL VEGF was added. Spheroids were
fixed after 48 h, and images were taken at 50magnification. VEGF induced strong sprouting, which was
completely inhibited by sunitinib. Scale bar 200μm
Fig. 3AMPKα1 is involved in VEGF-induced sprouting. HUVEC were seeded on 60-mm cell culture dishes and
transfected with 0.5μg/mL control-siRNA or 0.5μg/mL AMPKα1-siRNA, respectively, using the SAINT-sRNA
transfection reagent. After 72 h, spheroids were generated from control and AMPKα1-depleted cells,
embedded in fibrin gels and stimulated with 10 ng/mL VEGF for 48 h. After fixation of spheroids images
were taken. VEGF-induced sprouting was inhibited in AMPKα1-depleted cells by approximately 45% as
described previously [2]. Note that in the paper published by Stahmann et al. [2], the sprouting effect induced
by VEGF was lower. This is likely related to differences in growth conditions, especially in the applied serum,
and to an optimized protocol as described here. Scale bar 200μm
528 Katrin Spengler et al.