Method: How to measure extracellular ATP release in attached cells
1. Seed cells for overnight growing with a full (complete) culture media to achieve at least 100% confluence into 6-well plates at 37 ̊C, 5%CO2. Older cell cultures with high density of grown cells can provide higher ATP release. You need 1 control plate and 1 more plate for each shear stress duration (1min, 5 min, or 15 min etc.). To score ATP release of membrane-polarized cells, one can grow the cells onto inserts of semi-permeablemembranes.
2. Replace cell culture media in plates assigned for control and shear stress experiment with fresh full free media (1 mL per well) and incubate for 1 or 2hrs. in CO2-incubator at 37 ̊C, 5% CO2.
3. Collect the cell culture samples from each well (100 μL per well) and keep them on wet ice till conducting analysis.
4. Shake the cells at minimal speed (for example ~100 rpm or 200 rpm) at 37 ̊C, 5% CO2 for 1 min, 5 min or 10 min. Control samples are kept in CO2-incubator without shaking, and taken just for collecting sample. Our data shows that shear stress during 1 min, 5min or longer generate significant release of ATP, and probably other molecules such as cytokines (this was not tested yet by us).
5. Collect samples (repeat the step 3.) after performing shear stress from experimental and control cell cultures.
6. Analyze ATP concentration in samples with using Invitrogen™ ATP Determination Kit, Perkin Elmer’s ATPLite Luminescence Assay or any other reliable ATP assay kit.
7. Recalculate the concentration of ATP in the media according to volume of cell culture media in cell well (1mL for 1st sample collection, 0.9 mL after 2nd sample collection etc). Concentration of ATP should be normalized to the number of cells in the plate well or protein quantity in cell lysate from the same well.
If you read and use this text, you are welcomed to donate