--Verity Software House, ModFit for Proliferation offers online tutorials

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1 Customers may want to use another analysis software package with data collected on Attune. Reason for this include: they already have the SW and are confortable with using it, their institution may provide a license, analysis-only packages generally have more analysis features than acquisition+analysis software programs. These software programs are expensive and require additional training to use. Attune has an advantage because its software if Free, unlimited seats. Attune files are 24 bit, and compatibility requires more recent versions of these software packages: FlowJo: version or higher (PC), or version 9.0 or higher(mac) FCS Express: version 3.0 or higher good idea to download all updates that are free ModFit: version 3.2 or higher Kaluza: version 1.1 or higher VenturiOne: version or higher Following is a Guide to using Attune files in these 3 rd Party Software Programs 1. Verity Software House (ModFit for proliferation and for cell cycle) 2. Treestar (FlowJo) 3. DeNovo Software (FCS Express) --Verity Software House, ModFit for Proliferation offers online tutorials To use Attune files in ModFit For Proliferation Modeling (like CSFE or CellTrace Violet): 1. When the file is opened into ModFit LT, click the Convert button: 1

2 2. Then, choose the parameter(s) to convert to 4-decade log domain, and click OK : 3. The file should be ready to use in the Proliferation Wizard. --Verity Software House, ModFit for DNA Content Cell Cycle Analysis offers online tutorials 2

3 FlowJo from Tree Star Inc., There are on-line tutorials and webinars NOTE: Ensure that the most current version of FlowJo is installed prior to using Attune FCS files. NOTE: Attune FCS files from versions prior to the v1.2 release incorrectly store the data as signed integer. If autoscale is used for these files, data that exceeds 2^24 in value may be arbitrarily assigned a value of 2^32. It is recommended that a fixed scale is used limiting data to a range of +/- 2^23 (equivalent to 6.92 decades). NOTE: Any data that is 0 or negative will be displayed on the axis. Since area can be 0 and negative, there is a greater tendency to see this data piled up on the axis. In order to see the data correctly, the Bi-exponential transform will need to be used. The default 4.5 decade transform is adequate; but, increasing the number of negative decades from 1-2 and changing the width basis from 1-20 may be required. 1. To use Attune FCS files in FlowJo, simply drag and drop the file into the FlowJo sample list. 2. Double clicking on the sample will automatically open a SSC vs FCS plot by default. 3. Gate on the ROI 4. Open the ROI subset and select the appropriate parameters for the desired plot type. 3

4 5. To apply compensation to data collected in software versions prior to v1.2, drag the single color controls collected in the Attune software into the FlowJo sample list, and open the compensation editor (Windows-> Open Compensation Editor or Ctrl-E). Select the necessary controls for the desired parameters and ensure that the positive and negative peaks are correct by selecting the Wizard Graphs tab and reviewing the data. If the peaks need to be adjusted, open the appropriate data set (+/-) in the FlowJo sample list and adjust the gate. Before finalizing the compensation setup, review the data in the Wizards Graphs tab. To apply the compensation to your particular sample, drag the desired compensation matrix over the desired sample in the FlowJo sample list. 6. To apply compensation to data collected in v1.2 software, simply open the sample and when selecting the fluorescent parameters, choose the parameters that are prefixed with Comp. In the v1.2 Attune software, the Spillover matrix is embedded in the FCS file and the matrix can be read by the FlowJo Software. 7. To adjust the matrix: open the Compensation Editor; and edit the name of the Defined matrix. This will allow the user to edit the matrix manually. 4

5 --FCS Express, DeNovo Software You can download a free 30 day trial form their web site. There are also on-line tutorials and webinars offered. NOTE: Attune FCS files from versions prior to the v1.2 release incorrectly store the data as signed integer. If autoscale is used for these files, data that exceeds 2^24 in value may be arbitrarily assigned a value of 2^32. It is recommended that a fixed scale is used limiting data to a range of +/- 2^23 (equivalent to 6.92 decades). 1. To use Attune FCS files in FCS Express, create the desire layout and open the desired FCS file. 2. Set the range for the scatter plot from 0 to 2^23 (8,388,608) on both axis; set the resolution to 1024x1024 (not required); and, set the X-axis and Y-axis minimum resolution to auto. 5

6 3. For the fluorescent plots, set the range from to 2^23; set the X-axis and Y-axis minimum resolution to auto; set the scale type to hyperlog; and, set the hyperlog transform value to These values are recommended starting values. The range may require adjustment and the hyperlog may require adjustment for each parameter. The fluorescent channels may also be displayed in log, linear, and any other transform. NOTE: Displaying plots in log scale may be in appropriate since the log tranform cannot display 0 and negative values. As such, compensated data and parameters collected using the area measurement will only show data with values greater than 0. Using bounded gates may only represent a fraction of the actual data that may reside beyond the limits of the plot. 6