Date: Wed Feb 13 2008 - 16:41:37 EST
Hi Lori, I feel your pain - working with fish blood presents similar challenges. You may want to try hypotonic lysis using water and then re-establishing tonicity with e.g. a 10x PBS concentrate after RBC lysis is complete. I haven't used it much as I don't have the luxury of large blood volumes from most species I'm working with, but it works fairly well on trout, and WBC recovery is good. The method that works the best is described by Crippen et al. (2001) in The Journal of Aquatic Animal Health 13:234-245. Hope this helps. Cheers, Sean Taylor SCION 49 Sala Street Private Bag 3020, Rotorua, New Zealand Phone +64 7 343 5899 Mobile +64 27 292 0341 Facsimile +64 7 348 0952 www.scionresearch.com "RICE,LORI P" <lrice@ufl.edu> To 14/02/2008 10:22 Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> cc Subject lysing mouse RBCs Hi, I recently posted a similar inquiry and did not get any responses. We are also trying to get rid of RBCs in about half ml of whole mouse blood. The difficultly is getting rid of the nucleated RBCs, which are much more prevalent in murine blood than human and do NOT lyse. We have used PharmLyse instead of FACSLyse to avoid using fixatives. We also used the lysis protocol of eBioscience. We tried staining before and after lysis. All of these protocols resulted in a lot of residual RBCs and non-specific staining, as determined by using TER119 for RBCs and CD45 (we are interested in the TER119 neg/CD45 neg population). Out of frustration, we went back to Ficoll-Paque Plus, a product that was recommended to us for mouse blood. This removes more RBCs with less damage, but the results staining before or after Ficoll separation varies dramatically. Does anyone have any insight into this? If you are looking for the CD45 positive population, try the good, but expensive SpinSep kit from StemCell Technologies. Lori -- Lori Rice, Ph.D. University of Florida lrice@ufl.edu
