You have more options than you think 1-use PerCp cy-5.5 on CD34 an excellent fluorochrome 2-use Pe - Cy7 on CD34 this works well and in our hands give an excellent signal intensity but batch to batch compensation is required 3 -if you want to use an unlabelled or biotinylated antibody you can use a selection of anti mouse or streptavidin secondary antibodies that have quantum dots conjugated to them , you then go and choose whichever emission properties you want by selecting the relevant quantum dot and reduce the compensation required significantly we almost exclusively use quantum dots as a secondary reagent now because of the signal intensity and reduced spectral overlap, Good luck Ian Ian Dimmick Flow Cytometry Core Facility Manager North East England Stem Cell Institute Bioscience Centre International Centre for life Newcastle Upon Tyne NE1 3BZ UK Ian.Dimmick@ncl.ac.uk Tel 0044 191 2418831 Fax 0044 191 2418666 (mob) 0044 7970344823 -----Original Message----- From: Jos Bosch [mailto:j.a.bosch@bham.ac.uk] Sent: Thu 20-Dec-07 15:10 To: cyto-inbox Subject: CD34 CD133 Dear Flowers, Am setting up a panel on a 6-color FACScanto to characterize stem cells in peripheral blood, and running into the usual planning problem on what fluorochromes to assign to particular antibodies. The main hassle is caused by the fact that CD133 (miltenyi) and KDR (R&D) are only available in PE and APC, so have to work around these. To make a long story short, there are two options I seem to be left with: 1) Substitute CD34 PE (clone 8G12, BD) for PerCP. Arseniev (1999) concluded PerCP was not really bad, but not really great either. What is your experience?? Was unable find any systematic comparison in the literature that looked at PE-Cy7 or APC-Cy7. Does anyone have good validation data? 2) Alternatively, I can buy CD133 biotin and label it with FITC. Does anyone have good experience with that approach? What can I expect with regard to signal strength, background, and prep hassle? Your advise is very much appreciated! Kind wishes, Jos Jos A. Bosch, PhD Lecturer in Exercise and Behavioural Immunology School of Sport and Exercise Sciences University of Birmingham Birmingham B15 2TT United Kingdom T: (+44) 0121 4148105 F: (+44) 0121 4144121 http://www.sportex.bham.ac.uk/staff/boschjaReceived on Fri Dec 21 12:18:00 2007
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