ROS detection with dihydroethidium

[cardosopedro]

Hi.

I'm hoping that someone here can help me. I'm detecting superoxide production by measuring the fluorescence of dihydroethidium (DHE). I made a 1st try some days ago but the results are strange... I acquired 20000 events for every sample. Weirdly, the drug-treated samples (in which I expect to have ROS production) resulted in a histogram with a very small peak and a very disperse distibution of fluorescence at the left of this peak.

This is what I was doing:

1) After the desired time of incubation with the drug, I add DHE directly to the medium, which includes some death cells that dettached from the bottom of the well (30', 37º C);
2) I collect the old medium in order to take the death cells dettached from the bottom of the well;
3) I trypsinize the attached cells and mix them with the medium colected in point 2;
4) Then, I do two washings and it's ready for the cytometer.

However, this week, I tried to do this without collecting the medium as said in point 1. The result was that I didn't get all that disperse fluorescence at the left of the main peak. So, the reason causing this are the death cells in the medium (I thought this was the correct way to do it because I'm working with cell death...).

Now my question is: should or should not I collect the medium containing most of the death cells??


Can someone please help?