From: Bob Leif To: cyto-inbox Although, I agree with Mario concerning the present commercial instrumentation, there is a better long-term solution. Multianode PMTs and optical dispersive elements would be a much simpler, compact, and more flexible approach to the instrumentation. There has been significant work in Chemometrics on deconvolving spectra. Hopefully, the manufacturers will get the idea. The hardware parts are available at exhibit for the SPIE Photonics West Meeting 20-26 January 2001, San Jose, California USA (WWW.SPIE.Org). The scientific sessions at the BiOS part of the meeting, including the Advanced Techniques in Analytical Cytology session, should also be of interest. -----Original Message----- From: Mario Roederer [mailto:Roederer@drmr.com] Sent: Monday, October 16, 2000 11:39 AM To: cyto-inbox Subject: RE: best multicolor instrument? To those considering experiments utilizing more than 4-5 colors... Let us remember that instrumentation is only one of the three major hurdles that must be overcome to perform these experiments "routinely." The other two are software and reagents. The latter is perhaps the most difficult hurdle--since you can now buy the instrument as well as the software. I won't say much on the software (everyone knows my view by now), other than to say that your choice is extremely limited... there's really only one package that can handle the complex needs of multicolor experiments, from off-line compensation of all the different colors to keeping track of the dozens (if not hundreds) of distinct gates applied to a single sample. Reagents, however, represents a much bigger hurdle for the average laboratory. Currently, only about 6 colors are commercially available (FITC, PE, Cy5PE/PerCP/Cy5.5PerCP, Cy7PE, APC, and Cy7APC). For several of those colors, the availability of reagents is extremely low. And as you quickly discover, you simply don't want to do any second-step stainings if possible (there's your answer, Maciej), so having primary conjugates is extremely valuable. This means that your laboratory will need to learn how to conjugate reagents. Of course, there's nothing complex about this--it's pretty routine, and the steps are all layed out on my web site. However, it means investing in buying (or making) bulk quantities of purified antibodies, plus the fluorophores. And if you want to make tandems, then it's another level of complexity and reagent purchases. The major investment in reagents is not dollars (because, in the end, if you make them yourself you'll save a lot of money), but time. Someone has to learn how to do the conjugations, how to optimize them for your conditions, how to properly validate them, and how to database & track them (we have over 600 different conjugates in our refrigerator). So don't let an instrument manufacturer sales rep convince you that you can start doing 8-color experiments by buying their instrument. Before you decide to tackle such a project, realize that you will have to commit serious time to reagent manufacture & development. Even if you stick to commercially-available reagents, it will take some time to develop an understanding of the additional problems encountered by these experiments. Nicole Baumgarth and I recently published a paper in J. Immunological Methods (243:77-97, 2000), which covers some of these problems and presents quite a bit of useful information on setting up multicolor experiments. (I can EMail the PDF file by request). Finally, of course, you'll need to learn how to use FlowJo. mr
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