Mario, You seem to hold (and propagate) the mistaken belief that FlowJo is unique in its ability to compensate data offline - it's my pleasure to inform you that FCSPress can also do this. In fact you might find FCSPress useful when adjusting those niggly few combinations that FlowJo can't cope with (for instance separating a FRET signal from donor and acceptor signals where a dim/bright control isn't available). You can download a demonstration copy of FCSPress from my website (http://www.fcspress.com) if you're interested:-) Ray Ray Hicks Cambridge University, Department of Medicine, Level 5, Addenbrooke's Hospital Hills Road, Cambridge. CB2 2QQ. UK Tel +44 1223 330149 | email rh208@cam.ac.uk ----- Original Message ----- From: "Mario Roederer" <Roederer@drmr.com> To: cyto-inbox Sent: Monday, October 16, 2000 7:38 PM 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 |
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:35 EST