AMPK Methods and Protocols

9. Prepare a cell suspension of 37,500 cells/mL, and add 1.2%
   methylcellulose at a ratio of 5:1 to receive a cell concentration
   of 30,000 cells/mL. Mix carefully by inverting the tube.


10. Add 100μL of this cell suspension per well of a round bottom
    96-well plate using a multistepper pipette. Each well provides
    one spheroid (seeNote 26).


11. Incubate the plates for 24 h at 37C and 5% CO 2 to obtain
    spheroids.

3.1.3 Embedding and
Stimulation of Spheroids

1. Prepare fibrinogen solution 4 h before starting embedding of
   spheroids.


2. Harvest spheroids from the 96-well plates using a multichannel
   pipettor, and collect them in a pipetting reservoir (seeNotes 27
   and 28 ). Use a separate reservoir per treatment condition.


3. Transfer the spheroids into a 50 mL tube, and centrifuge for
   4 min at 200gat room temperature without brake (seeNote
   29 ).


4. Aspirate supernatant carefully to avoid damage of spheroids.
   Add 10 mL of HEPES/Ca buffer to the spheroids, mix care-
   fully by gently inverting the tube, and centrifuge again for
   4 min at 200gat room temperature without brake.


5. Calculate the amount of required thrombin (10μLof20U/
   mL thrombin is needed for each well with embedded spher-
   oids). Dilute thrombin stock solution (100 U/mL) 1:5 with
   HEPES/Ca buffer to obtain the final concentration of 20 U/
   mL (seeNote 30).


6. Aspirate the supernatant of spheroids carefully. Add 2 mL of
   the diluted fibrinogen solution (1.8 mg/mL) including apro-
   tinin to approximately 200 spheroids, and mix carefully (see
   Note 28).


7. Add 10μL of 20 U/mL thrombin per well of a non-coated
   24-well cell culture plate (seeNote 31).


8. Immediately add 300μL of the spheroid/fibrinogen suspen-
   sion to each thrombin-containing well of the 24-well plate, and
   gently pipette it up and down twice (seeNote 32).


9. Incubate the plate at 37C without CO 2 supply for 20 min for
   polymerization of fibrin (seeNotes 33and 34 ).


10. When the fibrin gel is formed, add 350μL of spheroid growth
    medium, and incubate for 30 min at 37C and 5% CO 2.
    Within this time, change the medium carefully after 10 and
    20 min without touching the gel (seeNote 35).


11. Aspirate the medium carefully and add 300μL of spheroid
    growth medium.

526 Katrin Spengler et al.