AMPK Methods and Protocols

3 Methods 3.1 Endothelial Cell Spheroid Assay 3.1.1 siRNA-Mediated Downregulation of AMPKα in HUVEC Seed HUVEC of the first pas ...

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 ...

Add compounds of interest (growth factors, drugs), and mix carefully by gently moving the plate (seeNote 36). Incubate the plat ...

3.2 Matrigel Plug Assay 3.2.1 Mouse Model Perform experiments with sex-matched 3-month-old wild-type and AMPKα1 knockout mice. ...

Cool all required solutions and materials to 4C (PBS, pro- and antiangiogenic compounds, 15 mL tubes, 2 mL tubes, pipette tips ...

3.2.3 Embedding of Matrigel Plugs in Paraffin Transfer the fixed plugs into labelled tissue cassettes. Labelling has to be perf ...

Wash the slides 8 min in PBS and incubate them for 8 min in PBST. Transfer the slides into an immunostaining system, and add PB ...

To obtain a representative evaluation for each plug, monitor two sections from at least four levels, respectively, and calculat ...

We obtain approximately 90% downregulation using AMPKα1- and AMPKα2-specific siGENOME SMARTpool siRNA from Dharmacon (GE Health ...

We use zinc fixative from BD Biosciences (BD 550523). Alter- natively, it can be prepared by adding 0.5 g/L calcium acetate, 5. ...

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

Inclusion of positive (for instance, bFGF) and negative con- trols (no treatment, inhibitors only) in an experimental series is ...

References Fisslthaler B, Fleming I (2009) Activation and signaling by the AMP-activated protein kinase in endothelial cells. C ...

Chapter 34 Analysis of Muscle Stem Cell Fate Through Modulation of AMPK Activity Marine Theret, Linda Gsaier, Sabrina Ben Larbi, ...

identified AMP-activated protein kinase (AMPK), the master met- abolic regulator of the cell, as an important player in MuSC sel ...

Polystyrene round bottom FACS tubes. 1PBS: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 8mMNa 2 HPO 4 ·7H 2 O, pH 7.4. Digestio ...

2.4 Immunolabeling of MuSCs 1PBS: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 8mMNa 2 HPO 4 ·7H 2 O, pH 7.4. Paraformaldehyde ...

Add 20 ml of DMEM/F-12. Mix by pipetting up and down. Filtrate the mixture on a 70μm cell strainer (seeNote 8). Wash the 30 ml ...

3.2 Measure of MuSC Proliferation Coat 48-well plate by adding 80μl of coating solution in each well and incubate for 30 min at ...

Change the media every day during 4 days (rinse once with DMEM/F-12 and then add fresh MuSC growth medium) and then every 2 day ...