Hi Juan,

How did you measured ROS ? using a Flow Cytometer or Plate reader ?

This is very common measuring ROS with DCF-DA like fluorescence probe using a plate reader because washing steps removes significant amount of apoptotic cells making the total number of cells in the apoptotic group is minor.

Though control cells produces relatively lesser amount of ROS, as their cell number is grater than in the apoptotic group, we get a higher fluorescence values.

This issue does not encounter with flow cytometer. If you still get them with the flow cytometer that's really interesting.

Cheers,

Susara

Dear Juan,

you have to check ROS accumulation through time (kinetics). It may happens that annexin-positive cells have much more ROS accumulation at early stages. Moreover, DCF-DA is not ratio-metric dye, thus, cant be used for quantification. One have to normalise it per dye uptake and conversion.

My best wishes!

Hi, Taras,

Thanks for your answer. I used CellRox Green to check ROS instead of DCFDA. Because I have read a review paper “

Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations” talking about apoptotic cells would release cytochromC and cytoC can also oxidize DCFDA to DCF then make the results not accruate.

Hi, Susara,

Thanks for your reply. I used CellROX Green followed by Flow cytometric analysis.

Thanks! Do not forget to use kinetics.

EOS accumulation come before Annexin activity. During and after may not correlate, because event already come (switch to apoptosis).

Best wishes!