How to setup gating for DNA fragmentation assay in Nucleocounter NC3000

Background

This DNA fragmentation protocol enables the user to quantify the DNA content of

mammalian cells in order to detect apoptotic cells with fragmented DNA (sub-G1 cells).

With DNA fragmentation assay, scatter plots and histograms are obtained from the NucleoView® NC-3000™ software. This document describes in detail how to set up gating to retrieve the number of cells with fragmented DNA in the sample.  

Gate setup for DNA fragmentation assay

  1. Right click on the sample to be analyzed and choose Show data. Different plots can be visualized by adding them to plot manager. Graph 1: dot plot for the DNA staining Area vs. Intensity, graph 2: histogram of DNA staining, graph 3: histogram of the DNA intensity. Row 1 is untreated control sample while row 2 is the treated sample with Camptothecin (CPT). 

2. Use the untreated control (row 1) to determine the Sub-G1, G1, S and G2/M phases. Double click on the histogram with the DNA intensity (graph 3). Add New Marker, M1 correspond to Sub-G1 population, M2 to G1, M3 to S and M4 G2/M phases and press OK. The percentage of the population in M1 to M4 is now shown in the graph.

3. Make row 1 as Master Row and click Apply, the same gating will be applied to the second row with the treated sample. 

4. To merge the histogram of the untreated and treated sample. First right click on the treated histogram (row 2) with DNA Intensity and choose Copy Histogram. Go to the untreated histogram (row 1) with DNA Intensity, right click and choose Paste Histogram.

5. The untreated sample cells with fragmented DNA (sub-G1) represent ~4% of the total cell population. In the CPT treated sample this percentage increases to ~60%. Right panel is a merge of the untreated and CPT treated samples (the red line represents the CPT treated cells).

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