You could overwrite the references. Or it may be better to add new tubes in the reference group so that you have all the options when unmixing the data. It's easiest to do this on the actual aurora - if you ever choose to do unmixing on another computer you'll have to move some instrument settings as well. Depending on how complex your experiment is, you might find it helpful to discuss with your Cytek Technical Applications Specialist to advise you on the best approach for your specific experiment.

@@UChicagoFlow Thank you for the stunning presentations! Please, how to transfer instruments settings between computers? I'm planning to unmix/analyze data on another computer to save machine time. Thanks!

@@pavlabohacova5906 You'll need administrative rights to make this change, so it may be best to talk to whoever maintains your Aurora. You'll need to transfer instrument settings using the data maintenance tool. Also my understanding is that you only need to do this if you're unmixing files that were recorded on different days. If everything was run on the same day you just need the fcs files/experiment.

Yes, it is possible. Just to make sure we're all on the same page: unmixing can be done in multiple applications (SpectroFlo, FlowJo, FCS express), though I personally find SpectroFlo the most user friendly. With SpectroFlo unmixing there is no way to view the "unmixing matrix". If you want to make a manual adjustment, you must use a separate method - compensation. Compensation can be done in multiple applications (SpectroFlo, FlowJo, FCS express) so that compensation is essentially applied on top of unmixed files. I would recommend manual compensation, not an automated compensation wizard. I am less familiar with FCS Express, but in FlowJo you must open the compensation editor and click "edit", which will generate and apply a new compensation matrix. To get a negative comp value you can select a box in the matrix and hit the down arrow key - typing a negative sign doesn't work but once you have a negative value you can manually type any value after the negative sign. And I feel the need to reiterate the most important point: manual compensation should be used with caution and for data that will be presented/published you are only allowed to adjust the compensation to correct the single stain controls and then apply that to the fully stained sample. Never ever adjust compensation directly on a fully stained sample.

When creating or editing your experiment, on the group tab select create or edit reference group, then in the window that lists all of the reference control tubes there is an "Add" button in the lower left corner that will allow you to add as many reference controls as you need.