The BD Accuri C6 uses fixed area scaling factors that do not normalize the area data values exactly to the height data values for all sample types. The reason for this difference is that the area data is displayed after area scaling factors have been applied. The difference between where the data is thresholded in Area vs Height will vary depending on the particles being analyzed. However, this may lead to a discrepancy in which the threshold falls when viewing Area data, as seen in Figure 2. There is no impact on the data if the Area parameter is viewed during data acquisition or analysis. All thresholds on the BD Accuri C6 are applied using the Height signal measurements. Typically, thresholds are used to exclude unwanted signals such as those from cellular debris. No ) Validation Beads can easily be visualized on an FSC vs SSC plot. In this example, is, therefore, also the trigger channel. On the BD Accuri C6, the default setting of a primary threshold on of 80,000 indicates that the system will record an event only if it has an FSC value of 80,000, regardless of any other parameter values. The signal pulse must exceed the level set on the trigger channel to be recorded as an event on any other channel. When using the BD Accuri C6, setting the primary threshold also defines the trigger channel. The trigger channel is the critical parameter used to determine if an event should be recorded. Thresholds A threshold is the lowest signal intensity value an event can have for it to be recorded by the cytometer. However, when viewing the Area parameters for the same sample (FSC-A vs SSC-A), the threshold appears to be lower than 150,000. In the vs plot, the data begins at 150,000. A sample of lysed and fixed human peripheral blood was acquired using an FSC trigger and threshold of 150,000. On the BD Accuri C6, the signal width is defined as the distance between the point at which the signal rises above the threshold and the point at which the signal falls below the threshold (time of flight). To be recorded, the pulse (black line) must have an intensity greater than the threshold value (red line).
2 The Noise Floor 4 Identifying Particles of Interest 4 Identification of Fluorescent Particles 5 Identification of Nonfluorescent Particles 5 Using Multiple Thresholds 5 Alternative Trigger Channels (Side Scatter and Fluorescence) 6 Data Acquisition Rate 7 Conclusion 8 Summary of RecommendationsĢ Signal Intensity Figure 1. This note discusses some of the basic concepts needed to perform small particle acquisition and analysis properly. 1 Threshold and Analysis of Small Particles on the BD Accuri C6 Flow Cytometer Contents 2 Thresholds 2 Setting the Threshold When analyzing small particles, defined as particles smaller than 3.0 µm, on the BD Accuri C6 flow cytometer, certain items must be considered to ensure proper data acquisition.
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