The BD FACSDiscoverTM S8 Cell Sorter acquires traditional fluorescent parameters as well as a set of imaging parameters. There are a handful of recommendations we can offer to make the use of this data easier in FlowJo version 10.10.
Preferences are accessed through the heart icon in the upper righthand corner of the workspace. The preferences for specific cytometers will be found under the Cytometers tab/button in the bottom row of options. To set the preferences for the BD FACSDiscoverTM S8, find the name in your list list of cytometers and select it.
The BD FACSDiscoverTM S8 fluorescent parameters will specify a Log transform function by default. We recommend checking the box for ‘Enable Transforms’ and setting a width basis of -100 to display the data on a biexponential scale, with a linear region of 100 units on either side of zero for generic scaling of S8 parameters, but an even better choice will be to use the advanced parameter scaling tool. Recommendations of specific values are below.
Advanced Scaling Recommendations
Default scaling can be assigned on a per parameter basis using the advanced parameter scaling preferences. Each of the six image derived parameters can be assigned a unique scale, forward and side scatter can be given different ranges, and the default range for fluorescent parameters can be aligned with the scale on the S8. Here we list scaling recommendations for S8 parameters that we at FlowJo have found useful.
| Parameter | Scale | Min | Max | Pos. Decades | Width Basis |
| Delta Center of Mass | Linear | 0 | 1.5 | N/A | N/A |
| Eccentricity | Linear | 0 | 1 | N/A | N/A |
| Radial Moment | Linear | 0 | 20 | N/A | N/A |
| Long/Short Axis Moment | Linear | 0 | 12 | N/A | N/A |
| Diffusivity | Linear | 0 | 120 | N/A | N/A |
| Center of Mass | Linear | 0 | 25 | N/A | N/A |
| Size | Biexponential | N/A | 10^3 | 3 | -75 |
| Max Intensity | Biexponential | N/A | 262144 | 4.5 | -100 |
| Fluorescent parameters | Biexponential | N/A | 8200000 | 4.5 | -100 |
| Forward scatter | Linear | 0 | 75000000 | N/A | N/A |
| Side scatter | Linear | 0 | 10000000 | N/A | N/A |
Some of these setting may vary for you data, particularly scatter if you are using larger or smaller cells, but you can visually adjust the scale while looking at the data using the advanced scaling tool if needed.
*IMPORTANT NOTE: Changes to Cytometers Preferences will only affect data loaded into a new workspace.
Parameter Filtering
We recommend using the parameter filtering options to filter out any parameter with -W, -H, or -T. These are each different methods of calculating a measure of fluorescent intensity from the measured electronic pulse. The -A parameter, which is the area of the pulse, is the most complete measurement, and -A is commonly the only measurement used for subsequent data analysis, making the other options redundant. Filtering them out pulse width (-W), height (-H), and time to peak (-T) can cut the displayed parameter list by 75%. However, alternate measures of some parameters can be useful for diagnostics and identifying single cells, so exempting the additional measures of side scatter seems useful and can be done by choosing ‘And not’ SSC.