Thursday, March 10, 2011

Just when I thought I was out...they pull me back in.

I'm speaking, of course, about BD and the FACSCanto platform. Now, if you know me and have talked to me about flow cytometers, you know I haven't been too kind to BD and the FACSCanto-A. We have, and continue to, battled with problems on these instruments. It doesn't help much that we have hundreds of users running all sorts of who-knows-what through the instruments. But then again, our LSRII's never break-down, our 15 year-old FACScan and 10 year-old FACSCalibur never break on us and they get just as much use. Recently, however, I happen to have the privilege of running a FACSCanto II with an HTS. We've had it in the lab for a few weeks, so I've been playing around with it trying my darndest to break it, without success. It's definitely not a fair comparison as far as the use and abuse our other cytometers deal with, but I've tried to run some unfiltered chunky samples on it a few times, and it recovers well. The HTS has been running as well as could be expected; so well, that we're now putting one on our Fortessa. There are two other things on the Canto-II that have really caught my attention. The first thing is the overall fluorescence sensitivity and resolution. It certainly ranks among the top instruments I've ever tested. I ran my standard battery of tests on the instrument (dim population resolution, linearity, dynamic range, and precision) and the FACSCanto-II excelled in all respects. I'll highlight a few of these tests with some figures below. Before that, I want to document just how awesome it is to be able to put on a regular 12x75 tube with about 100ul, and be able to analyze almost all 100ul out of the tube. The way that the lever pushes the tube all the way up so that the probe is nearly hitting the bottom is awesome. That, combined with the absence of a DCM sleeve on the SIT makes this possible.  Dead volume is about as minimal as can be for a tube loader.

Figure 1 below simply shows some antibody binding beads from Bangs stained with a CD4 antibody coupled to either FITC, PE, or eFluor450. The lowest stained peak represents a binding capacity of ~3000 antigen. To put that into perspective, CD4 on human Tcells is expressed at about 50,000 antigen. So, you can see that something with 10-fold less antigen density can easily be separated from background.

Figure 2 is simply displaying 8-peak bead data.

And Figure 3 is showing PI stained CEN's to show precision (% CV of the 1st peak) and linearity peak-to-peak ratios.