AMPK Methods and Protocols

33. Use an incubator without CO 2 supply or a heating plate at
    37 C to allow pH maintenance via HEPES buffer.


34. Control fibrin formation in parallel in a reaction tube. Dis-
    turbed fibrin formation may be due to (1) decay of fibrinogen,
    (2) insufficient activity of thrombin (repeated thawing/freez-
    ing, storage at room temperature), and (3) insufficient mixing
    of thrombin with the fibrinogen/spheroid suspension.


35. This step is required to equilibrate spheroids with medium and
    to remove residual thrombin.


36. The concentration of added compounds has to be calculated
    for a total volume of 600 μL per well (300 μL of fibrin
    gelþ 300 μL of spheroid medium).


37. The standard incubation time is 48 h. If sprouting is enhanced
    (seeNote 38) or spheroids are unstable, evaluation after 24 h is
    advisable.


38. Spheroids may exhibit high basal sprouting due to robust
    pipetting, insufficient removal of thrombin, transfection,
    and/or age of cells. In the latter case, incubation of spheroids
    in low serum conditions before embedding may be of use.
    VEGF-induced sprouting may be low when VEGF is inappro-
    priately stored or repeatedly thawed/frozen.


39. For storage, add the lid of the plate and seal the plate with
    parafilm. Plates with spheroids can be stored for approximately
    2 weeks at 4 C. In case longer storage is required, add
    0.2 mg/mL sodium azide. Note that prolonged storage will
    affect the quality of the spheroids.


40. For quality improvement of the pictures, remove the lid of the
    plate.


41. Take pictures from 5 to 10 representative spheroids per well.


42. Do not evaluate spheroids at the edge of the well because
    sprout formation may be spatially impaired. Use phase contrast
    in case spheroids appear too bright.


43. If the meta-file is read correctly, the program should give the
    length of the polylines inμm range (in average between 10 and
    100 μm per sprout).


44. Some Matrigel gets lost during pipetting due to its high vis-
    cosity; thaw about 30% more than the volume required for
    injections. Non-used Matrigel can be frozen again but avoid
    multiple thawing/freezing cycles.


45. Pipette tips are cooled by putting them into sterile tubes in an
    ice bath.


46. The Matrigel flask can be slightly rotated but avoid inverting it
    upside-down.

Angiogenesis 535