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 7.6.1 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 4.0.2 or higher
Following is a Guide to using Attune® files in these 3rd 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
http://www.vsh.com/ 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:
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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
http://www.vsh.com/ offers online tutorials
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FlowJo from Tree Star Inc., http://www.treestar.com/ http://www.flowjo.com/index.php
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.
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To use Attune FCS files in FlowJo, simply drag and drop the file into the FlowJo sample list.
Double clicking on the sample will automatically open a SSC vs FCS plot by default.
Gate on the ROI
Open the ROI subset and select the appropriate parameters for the desired plot type.
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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.
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--FCS Express, DeNovo Software http://www.denovosoftware.com/
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.
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3. For the fluorescent plots, set the range from -1000 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 2500.
4. 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.
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