ATP

Materials:

white costar plate (with or without glass bottom)

Cell-titer Glo PROMEGA (G7570, reconstitute as from datasheet, can freeze and thaw)

ATP for standard curve

Method

Per cell ATP quantification:

plate same number of live cells (trypan blue exclusion). Around 30,000 cells are in complete medium in 100µL fresh media.

Make blank with 100µL fresh media.

Make std curve diluitions (from 0.1pmol to 10,000 pmol in 100µL PBS per point).

Equilibrate RT

Add 100µL Cell-Titer solution

mix 2' on orbital shaker

let stand 10'

Read at Victor3 (3A2) luminNic program 0.4ms reading time.

Per protein quantification:

same as above but:

lyse cells, pellet debris, quantify BCA and use 250ng to 1µg of protein in 200µL PBS. Perform all steps at 4°C in ice and as quickly as possible.