1 h in 1% DMSO, may be toxic to the cells and lead to AMPK
activation even in vehicle controls. Compounds dissolved in
water, culture medium, or other solvents can also be used, as
long as the appropriate vehicle control is always included in the
experimental design.- To preserve activation and phosphorylation status of AMPK
and downstream targets, plates must not be removed from the
incubator for longer than necessary. To facilitate this, we
remove plates from the incubator and treat them in batches
of four, at intervals of 3–5 min. - Remove four plates from the incubator and place in the laminar
flow cabinet. Treat cells with compounds of choice. Swirl plates
gently to mix compound in the culture medium, and return to
incubator. Repeat at 3–5 min intervals until all cells have been
treated. - Lyse plates after an appropriate incubation period (for many
canonical activators, 1 h is sufficient), as described in Subhead-
ing 3.2.
3.2 Methods
of Harvesting Adherent
Cells for AMPK Assays
or Monitoring Thr172
Phosphorylation
- To preserve activation and phosphorylation status of AMPK and
downstream targets, cells must be lysed rapidly on ice. Indeed,
“slow lysis” at room temperature is a previously used method for
artificially activating AMPK (seeNote 8). For rapid lysis (lysing
four dishes at timed intervals), aspirate off the medium and then
gently but rapidly wash plates in 22 ml of ice-cold PBS,
aspirating the washing solution each time (seeNote 9). - Add 200–300μl of ice-cold lysis buffer; the exact volume
depends on the cell type but is designed to maximize protein
concentration in the lysate. - Immediately scrape lysing cells off using a cell scraper, and
transfer to prelabeled microcentrifuge tubes; vortex briefly
and flash freeze in liquid nitrogen. - Defrost lysates when required on ice, and clarify by centrifuga-
tion at 17,000gfor 10 min at 4C. - Remove and retain supernatant for subsequent protein quanti-
fication assays, Western blotting, and/or AMPK activity assays. - Cell lysates are stable at 80 C for many years. However, avoid
repeated freezing/thawing.
3.3 Methods
of Harvesting
Nonadherent Cells
(e.g., T Cells) for AMPK
Assays or Monitoring
Thr172
Phosphorylation
- After treatment, transfer cell suspension into a conical or
round-bottomed centrifuge tube on ice. - Centrifuge at 250gfor 3 min at 4C to gently pellet the
cells; remove culture medium by aspiration. Do not centrifuge
with higher g forces, as this may stress the cells,
activating AMPK.
Intact Cell Assays to Determine Contributions of AMP-Binding and ADaM Sites 245