Method: How to measure extracellular ATP release in attached cells
1. Seed cells for overnight growing with a full (complete) culture media with toachieve ~100% confluence into two 96-well, 24-well or 12-well plates (“I” and“II”) at 37 ̊C, 5% CO2. Alternatively, grow the cells in the inserts ofsemipermeable membranes.
2. Replace cell culture media with serum-free media (50μL per well for 96-wellplate, 200μL per well for 24-well plate etc).
3. Incubate the cells with serum-free media for 30 min.
4. Collect the cell culture samples from plates I and II and freeze themimmediately on dry ice.
5. Rotate (shake) horizontally on minimal speed (~200 rpm or so) at 37 ̊C, 5% CO2for 1 min.
6. Collect “stress” and “control” samples of culture media (50 μL per well for 96-well plate, 200μL per well for 24-well plate etc) with immediate freezing themon dry ice after collection.
7. Repeat rotation and collection samples with the same conditions for 5, 10 ormore time. Control samples are treated similarly but without horizontalrotation/shaking.
8. Analyze ATP concentration in defrozen (on wet ice) samples with usingInvitrogen™ ATP Determination Kit or Perkin Elmer’s ATPLite LuminescenceAssay