I'd like to image ROS accumulation in cells with a fluo dye and I'm wondering if anyone have ever performed cell lysis to mesure ATP after ROS imaging ? Does the ATP quantitation is still reliable ?
Hi Guillaume,
Unfortunately, it's practically unreliable assessing the ATP quantification in the cell lysates in which ROS accumulation was undertaken. Just compare the toughness of the later procedure, in order to imagine the inability of coexistence for delicate ATP reaction.
Intracellular ROS levels usually assess using the CM-H2DCFDA probe. Cells grown in 96-well flat clear bottom black polystyrene microplates are washed twice with 150 μL of HBSS to remove culture medium. Then, 50 μL of the probe (7.21 μM in HBSS) added to each well and the plates are incubated for 30 minutes at 37°C. Excess probe was discarded and 100 μL of the prooxidant solutions prepared in HBSS added. After the incubation, cells are lysed by adding 50 μL of 0.5% Triton X-100 in PBS. Cellular levels of ROS are visualized under the microscope using CM-H2DCFDA fluorescence live cell imaging; cell fluorescence images captured using an epifluorescence microscope.
At the same time, intracellular ATP determination performed by a bioluminescence assay based on the ATP-dependent luciferin-luciferase reaction. A new internal calibration standard of ATP usually prepared each day in a range from 1 to 100 μg ATP/mL prior to readings. To determine the cellular ATP content, cells grown on 96-well white clear-bottom plates were first incubated for 3 minutes with 25 μL somatic cell ATP releasing reagent and then for 3 minutes with 25 μL of sterile water. The plates placed in a Luminoskan RS luminometer and 50 μL of the luciferase-containing buffer (adenosine 5′-triphosphate assay mix) added to each well just before measurement of the light emitted, which is proportional to the ATP concentration.
Best wishes,
Ilya
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