HOWTO: Export FCS files

I’ve tried to make Cytoflow both powerful and straightforward to use, but I understand that some people may wish to use other software for their cytometry analysis. Cytoflow has some unique capabilities – in particular, its Bead Calibration and TASBE modules – and it is possible to import data into Cytoflow, calibrate or otherwise manipulate it, then export it back to FCS files that can be opened with other analysis programs.

Procedure

  1. Import your data into Cytoflow. Do not import your control samples (unless they’re part of the experiment.) In the example below, we’ll have three fluorescence channels – Pacific Blue-A, FITC-A and PE-Tx-Red-YG-A – in addition to the forward and side-scatter channels.

    ../../_images/bleedthrough2.png

  2. Perform whatever calibration or manipulation you would like. In this case, I’ve applied the Autofluorescence and Bleedthrough modules.

    ../../_images/bleedthrough7.png

  3. Choose the Export FCS view. It’s the FCS button. Note that this is found on the Views toolbar, not the Operations toolbar.

  4. Select how you’d like the data split up when it is exported. Remember, when Cytoflow imports data, it “forgets” everything about the “tubes” that the data came from – it only knows which sets of data were treated with which conditions. Select which conditions should be split into separate FCS files in the view parameters.

    ../../_images/fcs2.png

    In this case, I’ve asked for each unique value of Dox to go into its own FCS file. The table in the view pane will show you which files will be created.

    ../../_images/fcs3.png

  5. Click Export… and select the directory to save the FCS files to.