1/2/2024 0 Comments Flowjo flow cytometryCells containing 4n amounts of DNA have double the area and height values whilst the width is roughly the same as cells containing 2n amounts of DNA. Fortunately using the height or width vs area allows you to separate the doublets from the single cells containing 4n amounts of DNA. It is important to distinguish between single cells that have double the amount of DNA and doublets as they will both show increased fluorescence when stained with a DNA dye such as propidium iodide. You can use FlowJo to analyze all of your flow cytometry data - regardless of the cytometer used to collect your data files. Removal of doublets is also crucial in cell cycle analysis. FlowJo software is used for the analysis of flow cytometry data. Doublets have increased area whilst similar height to single cells. Doublets (red) can be distinguished from single cells (green) by plotting FSC height vs FCS area. Therefore disproportions between height, width and area can be used to identify doublets.įig. Doublets will have double the area and width values of single cells whilst the height is roughly the same. Schematic of processing of doublets.ĭoublet exclusion is performed by plotting the height or width against the area for forward scatter or side scatter (Figure 27). If a doublet containing a fluorescence positive and negative cell passes through the laser (Figure 26), it will produce a positive pulse leading to false positives in both analysis and sorting experiments.įig. Tens of thousands of cells can be interrogated by a flow cytometer in a single second. The cells flow single file past focused laser beams and light scatter or fluorescence data is collected. This can be critical in cell sorting, cell cycle and DNA analysis. Flow cytometry measures phenotypic, biochemical and molecular characteristics of individual cells or particles suspended in a fluid stream. Doublet exclusion as the name suggests, is to ensure you count single cells and exclude doublets from your analysis.
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