Hi Susan, I am a little confused by your indication that in order to get the beads on scale in the SSC parameter, the FSC is set too low to keep the cells on scale. FSC and SSC parameters are set separately so one should not affect the other. I have used Beckman-Coulter's Flowcount and BDB Trucount beads extensively and the FACSCalibur needs to be set up sightly differently to use these products properly. For example, by comparison with a Coulter XL or FC500, the FSC threshold needs to be lower on the BD instruments versus the Coulter ones, otherwise the Flowcount beads appear too small to be acquired. In the single platform ISHAGE protocols that are used on both instruments, Flowcount is detected on a plot of FSC versus time on the BD platform, whereas the beads are usually detected on an FL3 versus time plot on the Coulter platforms. The beads are generally 'off- scale' on the SSC parameter when the voltage is set to display leukocytes adequately, regardless of BD versus Coulter. I think you should consider using time as a parameter in your studies. These beads are usually found in the brightest FL1, FL2 and FL3 channels under 'normal' PMT settings. If you alter (lower) one of the PMTs of the BD instrument to see the singlet flowcount beads, you effectively render that channel pretty useless for the detection of anything else. We initially lowered to FL3 PMT on the FACScan in order to see the singlet beads. In sodoing, it was somewhat problematic to get viable (ie 7-AAD- cells) properly on scale in the same plot. Gating singlet beads (as one must since the assayed bead concentration is on the single beads) on the time versus FSC plot solved this problem completely. With Trucount beads, the assayed bead concentration is on the total (singlets plus aggregates). However, these beads are so small that NO forward scatter threshold can be used. A threshold must be set on another parameter, and for the Trucount version of the single platform ISHAGE protocol, this parameter is FL1. You can identify the Trucount beads on an FL1 versus FL2 plot and they are so bright that they cluster far enough away from other events that they can be easily identified and gated. HTH Rob Sutherland Room 11E 085 University Health Network/TGH TorontoReceived on Mon May 1 11:38:01 2006
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