To give an example of TO-PRO-3, see the attached image. In this case, the HV for the detector is a bit high since we're using the PMT for two signals: the 633nm line from the HeNe laser excites TO-PRO-3 and the 488nm line excites PerCP. This dye was not listed in my chapter on viability probes in Current Protocols in Cytometry pp. 9.2.1-9.2.14, 1997, but the balance will give an overview of various methods. Dave ============== David M. Coder, Ph.D. Director, Cell Analysis Facility Univ. of Washington School of Medicine 1959 NE Pacific Street H474A Box 357650 Seattle WA 98195 tel. 206-685-3014 email: dcoder@u.washington.edu ----- Original Message ----- From: Ray Hicks <rh208@cam.ac.uk> To: cyto-inbox Sent: Wednesday, February 14, 2001 3:20 AM Subject: Re: Viability with PI Hi Cheryl, I've seen the same things, and using PI in the same "channel" as another fluorochrome worries me too, although it's possible to mostly compensate it out of FL2 and use FL3 to discriminate live/dead . These intermediates are clearly visible in and annexin V versus PI apoptosis assay and are mostly annexin V high. I advocate the use of 7-AAD along with PE and FITC because it has less of a signal in FL2 even if it's not as bright as PI (Mario). If you've got the luxury of a red laser on your machine then you can use a red-excited dye such as to-pro 3 from molecular probes and have three untainted colours from your 488 laser (as suggested a few years ago on this list by Derek Davies). Cheers, Ray
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