- “Slow lysis” is performed at room temperature; this causes cell
stress, thus activating AMPK. Aspirate medium, add 500μlof
PBS stored at room temperature, scape cells off using a cell
scraper, and transfer to labeled microcentrifuge tubes. Centri-
fuge (17,000g, 3 min, room temperature), aspirate off the
supernatant, and wash the pellet once in 1 ml of PBS. Centri-
fuge again, remove the supernatant, and resuspend the pellet in
200–300μl of ice-cold lysis buffer. Samples can then be frozen
in liquid nitrogen and clarified as described in Subheading3.2,
step 4. - The PBS washes are essential to remove all traces of protein
derived from serum in the medium. - It is important when removing the top (aqueous) phase to take
only this layer. This means that a small amount of the aqueous
layer, and hence adenine nucleotides, may be lost with each
extraction. However, the losses will be the same for AMP, ADP,
and ATP, and if the results are always expressed as AMP/ATP
or ADP/ATP ratios, any losses during extraction will not
matter. - To determine the appropriate number of cells to use per well, it
is important to perform pilot experiments to find the numbers
of cells that give a linear relationship between cell number and
measured oxygen consumption rate. This will vary based on
the metabolic profiles of the cells used, but for HEK293 cells in
an XF24 analyzer, we would typically use 40,000 to 60,000
cells per well, giving around 70% confluence. - It is important to seed the cells in a low volume of medium to
ensure a consistent monolayer of cells at the bottom of
the well. - This helps promote even cell distribution across the well.
- For HEK293 cells, we use 25 mM glucose, 2 mM sodium
pyruvate, and 1 mM glutamine. - This volume of medium will depend on the number of injec-
tions to be performed. For an XF24 analyzer, we inject 75μl
from each port. The recommended capacity of the well is
900 μl, so for four injections, the cells are left in a final volume
of 600μl. - The XF24 can perform four injections on the same well, allow-
ing for increasing concentrations of the same drug to be tested
in the same experiment. It is also very informative to include
injections of known metabolic inhibitors to determine the
mode of action of test compounds. For example, we routinely
use 2,4-dinitrophenol (DNP) to test whether a particular com-
pound is inhibiting the mitochondrial electron transport chain
(ETC). The addition of DNP, which collapses the proton
Intact Cell Assays to Determine Contributions of AMP-Binding and ADaM Sites 251