Normalizing FCS Files - nolanlab/bead-normalization GitHub Wiki

Put the FCS files that you want to normalize in a folder that does not contain any other FCS files (other file types are okay).

Open the Normalizer.

Select the folder containing the FCS files that you want to normalize.

Select the set of masses that correspond to the beads you used.

If you forgot to acquire data on one of the bead channels, or have any other problem with a particular channel, you may choose 'Custom' and then select the subset of the bead masses that you want to include.

Adjust the bead gates for each file by dragging the corners of the boxes. If you cannot see clear bead populations in some of the channels, be generous with your gate on that channel, as only the events falling in the intersection of all the gates will get used.

Optionally, to see which events are in the intersection of your gates, press the space bar. The selected events will become red.

To move on to the next file, press Enter.

After you have gated the beads from all files, you will be asked whether you want to normalize to the median level of the current files, or to previously saved FCS files. Choose 'current files' if all the files you want to compare are included in your current normalization analysis. If you have already normalized most of the files in an experiment, but now want to add more and have them normalized to the same level, then select 'saved files.' You must then select the '_beads.fcs' files that were output when you normalized the earlier batch of your experiment.

Respond to whether you want to go through the bead removal step.

Either way, you should check your normalized files and consider doing a cleanup gate manually. If you are removing beads, enter the number corresponding to the color you want to use as a cutoff.

After you have cycled through all the FCS files, a plot will appear that shows the bead levels before and after normalization.

There is now a 'normed' folder that has been created inside your original folder which contains the normalized data. There is also a folder labeled 'beads' which contains both files of the unnormalized beads and files of all the events that have been removed.