OpenFlow: Full Spectrum Flow Cytometry with the Cytek Aurora

[@mariapardo9137]

Very informative, thank you very much from Panamá!

[@Runningchad2010]

Hello. Thank you for this presentation

[@siddharthmehra9226]

very informative and explained clearly thanks a lot

[@pennyanders4200]

Very informative! Thanks.

[@feitu6403]

A nice series of talks on the spectral flow cytometer. Here is my question for reference. The aurora allows us to re-use stored preferences for the same set of other experiments. I would like to know why this is allowed? When I do conventional flow cytometry, single stainings should be run every time before samples. Looking forward to your answer! Thanks in advance.

[@kathydaniels1077]

Hello Fei Tu. Thank you so much for a great comment. It is always best practice to run your single color controls and calculate unmixing at each run. There can be differences in instrument/reagent performance from day to day and the only way to account for this is through running single color controls. Thank you! - Kathy

[@feitu6403]

@@kathydaniels1077 I see! Thanks for your reply.

[@junaidrehmani9829]

How do we preemptively create a worksheet. I see you changed from default to openflow worksheet. Thanks

[@kathydaniels1077]

In any experiment you create, you can see that once the experiment is open there is an option next to the default raw worksheet with a little plus icon to the right of that. From there you should be able to add another worksheet and customize it as you see fit and then you can save this worksheet for the future which you can make your default worksheet in the acquisition module. I typically prefer to run an experiment first and unmix to set up my gating strategy with the unmixed results and then save that worksheet specific to my experiment so I can apply it to experiments in the future that follow that same heirarchy. Does that make sense?

[@feitu6403]

A question for creating a reference group at 23:57. Since there are several single stainings by beads and also a single stain by cells. Does that require an unstained bead group? In another way, if we use some single stainings by beads as well as other staining by cells, should we include two unstaining tubes, one for beads and another for cells? Thanks.

[@kathydaniels1077]

When mixing beads and cells, you do not necessarily need to run a separate unstained bead control. There is an option to have this unstained separate bead control in the software. If you do this, ensure that you specify in the setup that the negative reference for those samples is the unstained beads. If you do not do this, there is a negative/positive histogram gate in the unmixing setup that you can ensure has the correct gating. For this, make sure that your beads do have an internal negative control. It's also important to note that no matter what you will always need unstained cells to carry out unmixing. If you are working with multiple cell types with differing autofluorescence patterns, you will need to run an unstained for each of these and carry out group specific unmixing in SpectroFlo. Please let us know if you have any questions. Thanks! - Kathy

[@feitu6403]

@@kathydaniels1077 Appreciate your detailed explanation! -Fei

[@syedahafsaali5208]

What does autofluorescent positive and negative controls mean?

[@RuiGardner]

Hi Syeda. I'm not sure I understand your question. Did we state there were positive and negative autofluorescence controls somewhere in the video?

[@junaidrehmani9829]

01:07 I think Rui is referring to the autofluorescence within the single cell control of postive and negative sub-populations. If I understand it right?