AMPK Methods and Protocols

3 Methods

3.1 Endothelial Cell
Spheroid Assay

3.1.1 siRNA-Mediated
Downregulation of AMPKα
in HUVEC

1. Seed HUVEC of the first passage 1 day prior to transfection;
   cells should be 50–80% confluent on the day of transfection.


2. Dilute non-targeting or AMPKα-isoform-specific siRNA in
   HBS. Vortex SAINT-sRNA for 30 s and dilute it in HBS
   (1:5). Prepare siRNA/SAINT-sRNA complexes by gently
   pipetting diluted siRNA into diluted SAINT-sRNA and add
   4 volumes M199/HSA to obtain the final transfection solu-
   tion. Gently invert the tube, do not vortex. Usually, 2.5 mL
   transfection solution consisting of 1.25μg/250μL siRNA
   solution, 250μL diluted SAINT-sRNA and 2 mL M199/
   HSA is added to a 60-mm cell culture dish.


3. Wash cells twice with HBSS.


4. Remove HBSS and add the prepared transfection medium to
   the cells.


5. Incubate cells for 4 h at 37Cat5%CO 2 , and then add 2
   volumes of endothelial cell growth medium without removing
   the transfection medium (for instance, 5 mL growth medium is
   added to 2.5 mL transfection solution on a 60-mm
   culture dish).


6. Incubate for 48–72 h.


7. Check siRNA-mediated downregulation of target genes in
   Western blotting experiments parallel to spheroid assays.

3.1.2 Generation
of Spheroids

1. Wash siRNA-treated confluent HUVEC monolayers twice
   with PBS.


2. Add trypsin/EDTA (for instance, 0.5 mL per 60-mm cell
   culture dish) for maximal 3 min to detach cells.


3. Transfer the cell suspension into a Falcon tube containing stop
   medium (2 mL/10^6 cells).


4. Rinse the cell culture dish with stop medium (2 mL/60-mm
   dish), and pool the rinsing solution with the initial cell suspen-
   sion (seeNote 23).


5. Centrifuge the pooled cell suspension for 6 min at 500gat
   room temperature without brake.


6. Aspirate the supernatant, and resuspend the cell pellet in 5 mL
   of endothelial cell growth medium (seeNote 24).


7. Take an aliquot of the suspension, and count the cells (for
   instance, using a Neubauer chamber).


8. Calculate the number of spheroids and the cell number
   required for the respective experiment. Perform each treatment
   condition in duplicates. One spheroid should contain 3000
   cells; approximately 30–40 spheroids per sample have to be
   planned (seeNote 25).

Angiogenesis 525