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UCFlow: Gating Annexin V data appropriately

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My 3-step approach to gating Annexin V data appropriately

Using flow cytometry to assess cell death, and more specifically, apoptosis, is an everyday occurrence in a flow core. Technically speaking, performing the assay couldn't be easier when utilizing numerous kits available from any number of vendors.

It's typically set up in the format, add 'x' ul of reagent A to your cells, then 'x' ul of reagent B, and analyze.  So, with such a simple assay, what could possibly go wrong?... Lots!

Let's assume there are no faults with setting up the instrument or parameter voltages and the like. Let's also assume the assay worked; that is, control cells looked relatively live, and treated cells looked nicely dead. The possible problem I've been coyly hinting at therefore, is in the analysis.

What's typically the first thing someone does when they analyze their flow data?  They make a forward scatter (FS) versus side scatter (SS) plot and gate on the 'live' cells. Obviously, if you're trying to determine frequencies of live and dead cells, you wouldn't use a 'live' gate, you'd extend the gate lower on the FS parameter to include the dead cells. However, when you do so, you could be including small debris and bits of cells, and it's not exactly fair to count fragments of cells as events. So, herein lies the dilemma: You'd like to count whole cells, while drawing a light scatter gate that encompasses both live and dead/dying cells. Hence, My 3-step approach to gating Annexin V data appropriately.