The relationship between the DNA histogram and the cell cycle is illustrated below. The figure does not show the cells in G0 (out of cycle). The G0 is shown in the histogram from the NC-3000 software, if it is of interest to the customer.
How To Place Markers
- Start by marking your G0/G1 cells. Click on New Marker at the top of the histogram and click once on either side of the G0/G1 peak. Make sure that your M1 bar is only as wide as the widest part of the peak.
- Mark your G2/M cells next, again the M bar should only be as wide as the widest part of this peak.
- Everything between M1 and M2 should now be S phase cells. The start of the M3 bar should be exactly where the M1 bar ends and should end exactly where the M2 bar starts.
- The Sub-G0 M bar is entirely optional and is normally not shown on cell cycle analysis data. It is shown here for informational purposes only. Sub-G0 is for those interested in the amount of apoptotic/ necrotic cells and debris in the sample. As cells die and undergo apoptosis, DNA fragmentation occurs. This can manifest itself in the cell cycle histograms as a population to the left of the G1 peak.
- Give your Markers a name by Selected Gate option at the top of the histogram.
How To Clean Up Data
You can also get rid of the sub-G0 peak by gating out the debris. The first row is the control and doesn’t have many events that are Sub-G0. However, row 2 there is a significant sub-G0 peak. In row 3, the debris have been gated out of the histogram and remaining peaks look more defined.
To do this, I created a DNA scatter plot by clicking on the bottom left button to make the DNA scatterplot. The P1 gate was created by pressing New Polygon on the scatterplot and drawing around the debris. On the histogram, select Gating Setup and then select P1, Show and Exclude (example below).
A table plot can be added to display the markers.