- We use zinc fixative from BD Biosciences (BD 550523). Alter-
natively, it can be prepared by adding 0.5 g/L calcium acetate,
5.0 g/L zinc acetate, and 5.0 g/L zinc chloride to 0.1 M Tris
buffer, pH 7.4. - Use serum from the same species as the secondary antibody
source. - BSA-c is a 10% (w/v) solution of acetylated BSA, which pre-
vents charge-based background by blocking polycationic sites
of specimen. - We obtain good results with the polyclonal anti-CD31 anti-
body from Acris (AP06560PU-N). - We use an Alexa-Fluor 488-labelled goat anti-rabbit secondary
antibody. - Prepare pooled cell suspension for each experimental condition
(i.e., siRNA treatment). - Suspend cells in 1 mL of growth medium before adding the
remaining 4 mL. - Embed approximately 20–30 spheroids per well of a 24-well
cell culture plate. Calculate for 30–40 spheroids since losses
occur during washing procedures. Higher numbers of spher-
oids per well may limit space for individual sprouting and lead
to fusion of adjacent sprouts. Run positive (treatment with
basic fibroblast growth factor (bFGF)) and negative controls
(no treatment). - To prepare 96 spheroids in a 96-well plate, mix 8 mL of cell
suspension with a concentration of 37,500 cells/mL and 2 mL
methylcellulose. - Check spheroids under the microscope before harvesting.
Instability may be related to poorly dissolved methylcellulose. - Use pipette tips with a sufficiently wide diameter at the end to
avoid damaging spheroids due to shear stress. - Do not pool spheroids from more than three 96-well plates to
restrict shear stress during future pipetting. - Dilution of thrombin is preferentially done during the last
centrifugation of spheroids. - Add thrombin as a drop in the middle of each well. Thrombin
will convert the subsequently added fibrinogen into a solid
fibrin gel. - Mix gently to obtain homogeneous fibrin formation. Avoid the
formation of air bubbles. Control each well under the micro-
scope for the number of spheroids and equal distribution. If
required, prepare additional wells from the residual spheroid
suspension.
534 Katrin Spengler et al.