Dear Flowers, I'm designing an experiment on a Coulter XL (FL1-4 from a 488 laser) and would like to measure calcium flux (Fluo -4 and Fura Red) in activated T cells (2 cell surface markers CD25 & CD69). After reviewing the previous listings on calcium/Fluo measurements by flow, I have the following questions: 1. It appears that most people use FL3 to measure Fura Red, which happens to fall right in the middle (597) of my FL2 (PE or 575) and FL3 (PE-Texas REd or 620). How big of an issue is compensation? 2. It also appears that most users recommend using the linear scale for the calcium measurements. If my FL1 and FL3 are linear, how do I compensate FL1 and FL3 with FL2 and FL4 if they are log? 3. How much of any effect will staining with cell surface markers (CD 25, CD69, or CD4, CD8, etc) have on calcium flux? Thank you in advance for your reply. Regards, David ********************************************* J. David Holtzclaw, Ph.D. NASA Johnson Space Center SK3 - Bldg 37 2101 NASA Parkway Houston, TX 77058 (281) 244-0988 (Voice) (281) 483-3396 (Fax) Email: david.holtzclaw1@jsc.nasa.gov <mailto:david.holtzclaw1@jsc.nasa.gov> Website: http://www.dsls.usra.edu/holtzclaw.html <http://www.dsls.usra.edu/holtzclaw.html> or http://userwww.service.emory.edu/~jholtzc/ <http://userwww.service.emory.edu/~jholtzc/> ******************************************** <<HOLTZCLAW, DAVID (JSC-SK) (USRA).vcf>>
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