Date: Thu Mar 22 2007 - 04:59:49 EDT
Hi B., Standard FACScans and -Caliburs measure only pulse height, which is why parameters were displayed as "FL1" etc, rather than "FL1-A" and so on. There was an option for "doublet discrimination" based on pulse processing in the old days, if I remember correctly only on the 'Calibur. Though I never got my hands on such a machine myself, I seem to recall from reading specs they didn't do that the way a present-day digital machine will (H, A and W), but rather standard pulse height (H) measurement and width (W). I may be off here on the details for lack of hands-on experience, but the bottom line is that most likely your 'Scan doesn't do pulse processing beyond pulse height measurement. Trying to get plots using parameters which aren't acquired will get you... empty plots! Why CQ will allow you to check the boc when the machine doesn't have the electyronics to do so is probably a user interface issue, unless I was wrong about 'Scans not having the DDM option. Guy Next generation therapeutic <http://www.ablynx.com/> antibodies Guy Hermans, PhD Senior Scientist Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: Lewis, Benjamin - lewby002 [mailto:Benjamin.Lewis@postgrads.unisa.edu.au] Sent: Wednesday, March 21, 2007 3:03 AM To: cyto-inbox Subject: Measuring FL-A and FL-W Hi all, I'm working on DNA content analysis with cancer cell lines treated with a particular apoptosis/anti-proliferative agent. I'm staining the (fixed) cells with 7-AAD as a means to find cell cycle block and apoptotic populations, working on a BD FACScan cytometer (Cellquest Pro software). To gate out doublets I'm setting a dotplot of FL3-A vs FL3-W, however this is where things go wrong. No events are represented on this graph, however there definitely is FL3 signal as I am getting FL3-H data. It just doesn't seem to be measuring FL-A or FL-W, or if it does it is too low to register on the scale. No matter what I've tried events don't register on the A vs W plot, I've tried changing gains, voltage, high flow rate, low flow rate, anything I can think of. (On the software the DDM box is ticked, and set to FL3 by the way). Our resident experts don't know as they haven't had to use A or W before, so I'm kind of stumped. Its not only FL3 that won't measure A or W as I tried PI on FL2 and the same occurred. I suppose for starters, is the BD FACScan actually capable of measuring FL-A and FL-W? I think it is as the user books that came with is talk about A vs W plots. If so, is there a set up step I'm excluding? All I'm doing is a new dotplot of FL-A vs FL-W, DDM box is ticked (supposedly to allow FL-A and FL-W measurement) and set to FL3 and thats it. (Obviously FL3 voltage is set to place G1 population on an FL3-H histogram) Any help would be very much appreciated. I realise this is a kind of difficult problem to explain so my apologies if it doesn't make sense. Thankyou Benjamin Lewis, BBiotech (Hons) PhD candidate Sansom Institute University of South Australia Reid Building, Frome Road Adelaide SA 5000 Phone 8302 2421 Benjamin.Lewis@postgrads.unisa.edu.au ----------------------------------------------------------------------- THIS E-MAIL MESSAGE IS INTENDED ONLY FOR THE USE OF THE INDIVIDUAL OR ENTITY TO WHICH IT IS ADDRESSED AND MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM DISCLOSURE. If the reader of this E-mail message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately at ablynx@ablynx.com. Thank you for your co-operation. "NANOBODY" and "NANOCLONE" are registered trademarks of Ablynx N.V. -----------------------------------------------------------------------
