How to Gate GFP Assay

Background

If the GFP is sufficiently expressed, you can detect this using the GFP module.  For poorly expressed GFP, you can try to detect this using FlexiCyte and increasing the exposure time.  

How To Gate GFP Assay

Raw Data.  Notice that the data is scrunched against the axes. In the first scatter plot, bring all the events into view by clicking on the axes and choosing either log or bi-exponential.  Whichever scale brings all the events clearly on the plot.

Here, the first scatter plot is now displaying all the events clearly.  The next step is to check all the axes in a similar manner.

Double click to open the second scatter plot and new quadrant at the top.  To help you decide where to put the quadrant you can check where the GFP positive cells are lying.  You can do this by clicking on a green cell in the image, whilst holding down the control button and a yellow square should appear.  Now navigate back to the plot manager and find the little purple cross.  You can also draw a New Polygon around some events, right click over the polygon and ‘add cells inside gate to image overlay’ and check the fluorescence on the image.

In the histogram, it is useful to place a new marker.  If a negative control were available, this would be best for finding out where to place the M1 marker bar.

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