How to Gate Mitochondrial Potential Assay

Background

The mitochondrial potential assay is a well-known, early apoptosis assay.

How To Gate Mitochondrial Potential Assay.

Adjust the axes in the first scatterplot so that all the events are displayed and not scrunched up against them.  Healthy cells are stained red so in the control, so the majority of events will be at the top of the scatterplot.  Next select New Quadrant and place it in the scatterplot.   The downward shift to green as cells go through apoptosis is not huge, so place the quadrant close to the main population. 

Next select New Marker and place it over the healthy Red population.  Now make Master Row 1 to duplicate the gating strategy in the treated sample.

Use the treated sample to guide you where to put green marker.  Make Row 2 Master Row.

The data should look something like this now.  You will notice that the numbers don’t really match up between the different ways of looking at the samples.  Now we need to tweak all the gates so that the data matches.  For example, in the control scatterplot there are 10% ‘green’ events which doesn’t correspond to the Green JC-1 histogram next to it.  Below the numbers match better.

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