Dear Ursula, The laser and the filters in the FACScan/Calibur will let you down I'm afraid - Indo-1 requires UV excitation to generate its violet and blue-green signals for ratioing. The usual strategy for 488nm work is to load the cells with Fluo-3 (or similar), and ratio the increase of green fluorescence against either the fixed red (isosbestic point) fluorescence of SNARF-1 or the decreasing red fluorescence of Fura-Red. It's best to switch time on as a parameter, although you can plot your ratio against a "calculated" time parameter based on the order in which the events are saved (naturally, this presumes an even and un-interrupted flow rate). At this point I'd recommend my program FCSPress for your kinetic analyses, it's very straightforward to use and it allows you to plot a variety of statistics against time including mean, median and percent responding (it even inserts time points automatically if you didn't acquire with a time parameter) - and you'll find that you can transit to it quite rapidly. You can see an example kinetic plot (and other plots of a calcium experiment at http://www.fcspress.com/html/FCSPressFeatures/graphics.html). You can download a copy from http://www.fcspress.com which will run for thirty days. Ray Ray Hicks 134 High Street, Harston, Cambridge. CB2 5QD. UK e-mail RayH@FCSPress.com Web http://www.FCSPress.com Tel +44 797 453 8647 Fax +44 870 740 8595----- Original Message ----- From: "Ursula Esser" <uesser@UCDAVIS.EDU> To: cyto-inbox Sent: Wednesday, December 27, 2000 8:25 PM Subject: Calcium flux experiments with standard BD Facscan/Facscalibur (instrument filters)? | | Dear Flowers, | | It's the holidays, and I have a hard time tracking down if I can do calcium | flux experiments (using Indo-1 AM or alike) by flow using the standard | equipped BD Facscan or Facscalibur instruments (in particular: filters ok?). | | As I have only performed steady state experiments, I am also not sure if I | have to change the acquisition mode in order to allow analysis using time | as a parameter (either in CellQuest or FlowJo (to which we are slowly | transitioning)). | | Any suggestions, hints, info, recommendations for data analysis? | | Thank you all in advance for your help. | | Ursula Esser | | -- End -- |
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:15 EST