(b) For a 6 cm plate, add 150μl of ice-cold 5% perchloric acid,
scrape off cells using a cell scraper, and transfer the liquid
into prelabeled 1.5 ml microcentrifuge tubes.
(c) Vortex for 30 s, and store on ice until all plates have been
lysed. Cold perchloric acid extracts are stable for 1 or 2 h
without significant degradation of adenine nucleotides,
althoughsteps 3to 6 below should always be performed
as soon as possible.- Clarify the lysates by centrifugation at 17,000gfor 3 min at
4 Cand transfer thesupernatanttoafresh microcentrifugetube. - Add an equal volume of a 1:1 mixture of tri-n-octylamine and
1,1,2-trichlorotrifluoroethane to the tube, vortex for 30 s, and
repeat centrifugation. - Carefully remove the top (aqueous) phase, transferring it into a
fresh tube (seeNote 10), and repeat the extraction by adding
an equal volume of a 1:1 mixture of tri-n-octylamine and 1,1,2-
trichlorotriflouroethane, vortexing for 30 s, and repeating
centrifugation. - Carefully remove the top (aqueous) phase, transferring into a
fresh tube, and store frozen for subsequent analysis. The
extracts should now have a neutral pH with no perchloric
acid remaining, and in this form, the nucleotides are stable at
80 C for many months. - Capillary electrophoresis (CE) detects nucleotide peaks by UV
absorbance at 254 nm. ADP and ATP levels are measured using
peak areas and expressed as ADP/ATP ratios. - The capillary is washed as follows: Buffer A 1 min, 20 psi; water
1 min, 20 psi; Buffer B 2 min, 20 psi; and Buffer C 2 min,
20 psi. The nucleotide sample is injected at 1 KV under reverse
polarity and chased by an injection of Buffer D for 1 min at
0.5 psi. Nucleotides are separated over Buffer C by reverse
polarity at 7 KV for 10 min. - The LC-MS column is maintained at a controlled temperature
of 30C and is equilibrated with 10% Buffer B for 5 min at a
constant flow rate of 0.06 ml/min. Samples are loaded onto
the column and compounds eluted with a linear gradient of
10%–60% Buffer B over 9 min. Buffer B is then increased to
100% within 1 min and the column washed for 5 min with
100% Buffer B. Eluents are sprayed into the TSQ Quantiva
using an Ion Max NG ion source with ion transfer tube tem-
perature set to 350C and vaporizer temperature 125C. The
TSQ Quantiva is run in negative ion mode with a spray voltage
of 2600, sheath gas 40, and aux gas 10.
Intact Cell Assays to Determine Contributions of AMP-Binding and ADaM Sites 247