This app is designed to quickly write your calibrated fluorescence parameter to the .fcs file. Writing this new parameter to the .fcs files also means that when files are shared on websites such as FlowRepository, others do not have to perform gating and apply regression to your files to view your data with MESF units.
Please contact me if you have any issues using the software or if there are any additions that would be useful in future versions.D
Checklist for Performing MESF Regression:
- MESF Beads (Bangs Labs Link, BD Link)
- Sample for calibration analyzed at the same voltage as the fluorescence channel the MESF beads were acquired on.
- Flow Cytometry analysis software to perform gating on the MESF beads and acquire statistics (Geo-Mean/Median) of each bead population
Steps to use:
1. Gate around MESF Bead populations
2. Obtain statistics for each of the beads (Geo-Mean/Median).
3. Open MESF Calculator application.
4. Input the MESF bead values and corresponding bead statistic in the ‘MESF Values’ and ‘Channel Number’ box and click ‘Add’.
5. Repeat for each of the bead populations. The Bead Fluorophore and what you would like to name the new parameter in the .fcs file.
6. Click ‘Menu’ then ‘Load FCS Files’.
7. Navigate to the folder containing the fcs files for which you would like to convert the axis to MESF units and click ‘Open’. The .fcs files will then appear in the FCS Filenames listbox.
8. Click the ‘Channel to Convert’ dropdown menu. This contains the parameters that are common to all of the loaded .fcs files. Select the channel that will be converted to fcs units and give the new parameter a name.
9. Click the ‘Perform Regression’ button. A figure will appear showing the regression curve. The equation will also now appear in the ‘Regression’ box in the application window. This equation can be copied directly into the FlowJo ‘derive parameter’ window as in MESF Calculator v1, if desired.
10. Click the ‘Write to FCS’ button in the bottom write to convert the selected channel to a new parameter in the fcs file that will appear by the name given in the ‘New Parameter Name’ box. The files will appear in a folder called ‘MESF_Export’ in the same folder the fcs files were imported from. The file directory the folder is saved in is also shown at the bottom of the MESFPASSwindow.
11. Import the files into analysis software of your choice, the files will now contain the converted MESF channel.