Friday, April 16, 2010

ISX Fluorescence Intensity versus FCM

One of the first questions people ask me about the ISX is how does the fluorescence intensity compare to a standard flow cytometer (FCM). In a quick attempt to partially answer that question, I ran some BD Calibrite beads on our LSRII-Orange and our ISX, and have posted the results in the attached slide. I ran PerCP beads in hopes of demonstrating that the high laser power and long illumination times on the ISX would photobleach the signal (which it did). I also ran FITC, PE, and APC beads as sort of a general comparison of commonly used fluors. The results are fairly nice. FITC is nearly identical on the 2 instruments, PE was brighter on the FCM (probably due to filter ranges), and APC was really bright on the ISX (due to the zero background fluorescence in this channel). The other thing to test was how well the IDEAS software compensated the spillover. For FITC and PE, it was fine. However, because the PerCP was so dim in the PerCP channel, (APC was actually brighter in the PerCP channel than PerCP was) compensating those two took quite a bit of trial and error on my part. I manually adjusted the comp values until it looked acceptable. The resulting plot pretty much explains it all. You'd be hard pressed to easily identify APC, PerCP double positives in this example (luckily there were none, so no problem). Anyway, just wanted to give you a quick peak into how things may match up when trying to set up your color choices for the ISX.

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