AMPK Methods and Protocols
- We obtain approximately 90% downregulation using
AMPKα1- and AMPKα2-specific siGENOME SMARTpool
siRNA from Dharmacon (GE Healthcare).
- The transfection reagent SAINT-sRNA (formerly SAINT-
RED, Synvolux Products and Therapeutics) uses the principle
of lipofection and shows good results in HUVEC.
- Autoclaving is required for sterility and does not affect the
structure of methylcellulose.
- Initial dissolving at 60C is critical; methylcellulose will not
dissolve at lower temperatures and will precipitate at higher
temperatures. Use a water bath rather than a heating plate.
Warm up the methylcellulose solution at 60C in a water
bath for 20 min, and take it out four to five times for 1 min
of stirring on a magnetic stirrer. The final overnight stirring at
4 C will lead to complete dissolution.
- Use only the upper 45 mL from 50 mL. The sediment may
contain residual cellulose fibers.
- Prepare fresh methylcellulose stock solution regularly; it should
be stored not longer than 6 months.
- HEPES buffer cannot be autoclaved since it contains glucose.
- Aliquots can be stored at 20 C for up to 1 year. Avoid
repeated thawing/freezing.
- Prepare approximately 200μL of the fibrin stock solution per
sample. Some of the fibrinogen is lost during dissolving, and an
extra volume may be needed for additional wells (seeNote 32).
- U-shaped tubes provide a larger contact area between fibrino-
gen powder and HEPES buffer.
- By adding HEPES buffer to the rim of the tube, you can wash
away the fibrinogen powder on the wall. Pipetting the buffer
directly onto fibrinogen leads to clumping.
- Protein determination can be done according to Lowry or
Bradford. Perform measurement at least in triplicates.
- Aliquots can be stored at 20 C for up to 1 year. Avoid
repeated thawing/freezing. Keep thrombin aliquots on ice
during use.
- Low serum concentration in spheroid growth medium is
required to reduce spontaneous sprouting.
- We use Matrigel from BD Biosciences (BD 356231), which has
a protein concentration between 9 and 12 mg/mL. Since the
composition of Matrigel batches may vary significantly, it is
essential to test the batch in a small pilot study before
performing a bigger experiment.
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