- Add compounds of interest (growth factors, drugs), and mix
carefully by gently moving the plate (seeNote 36). - Incubate the plate with spheroids at 37C, 5% CO 2 for 24 or
48 h (seeNote 37and 38 ).
3.1.4 Fixation
and Analysis of Spheroid
Sprouting
- Fix the spheroids 24 or 48 h after stimulation. Carefully aspi-
rate the medium without touching the gel, add 500μL cold 4%
PFA to each well, and incubate for 10 min on ice. Aspirate PFA,
wash the gel twice with 500μL of PBS, and add 500μL of PBS.
At this stage spheroids can be stored (seeNote 39). - Take 50magnification pictures of the fixed spheroids in
bright-field microscopy using an inverse microscope (see
Notes 40– 42 ). Save TIFF-files and corresponding meta-files. - For analysis, load the TIFF-files into a software, which is able to
process meta-files and to acquire the correct scale information
of the picture. - Determine the number and length of sprouts per spheroid. For
this, zoom into the pictures, and trace the sprouts from their
origin to the end using the “polyline” tool of the software (see
Fig. 1). Export data on the number and length of polylines
(i.e., sprouts) into a spreadsheet program (seeNote 43). Cal-
culate mean values of sprout number and length per condition. - Calculate mean values from several independent experiments,
and perform statistical analysis (representative images shown in
Figs.2 and 3).
Fig. 1Analysis of sprouting from HUVEC spheroids. (a) TIFF-files of spheroid images (50magnification) are
loaded into an appropriate software for quantification. (b,c) Spheroid sprouts are traced with the toolpolyline
from their origin to the end including kinks. (d) Data are exported into a spreadsheet program. The number of
polylines and their length inμm correspond to the number and length of the sprouts. Five to ten spheroids per
experimental condition are quantified and averaged. Mean values and standard deviations need to be
calculated from several independent experiments. Scale bar 200μm
Angiogenesis 527