At 18:20 11/19/97 +0000, Dr Vishal Kumar Gulati wrote: >I would be grateful if people on this list would like to share their >experiences about FACsing fibroblasts and hepatocytes. Vishal: I like to think I can help... The obvious: trypsin is out. You'll have to use a EDTA/PBS solution to get the cells off. I have always used 5mM EDTA in PBS. You could also seed them on a layer of collagen and use collagenase in hopes of getting them off in single cells - I've never tried this as of yet. No matter what, you will get doublets, triplets, etc., going through the machine in addition to just "singles". Having just cleaned a massive clog from my 70uM Vantage nozzle, I'd advise a pre-filter of 60 uM before running! The EDTA solution causes some mortality. In my hands, it is proportional to how tenacous the cells are in clinging to the flask in the EDTA solution. COS and HEK cells, not long as they come off fairly quick. However, epithelial cell lines and HepG2 cells, can take upwards of 10 minutes before they "decide" to come off. Keritinocytes (from the next lab, I'm told) can take as long as 25 minutes before they come off. You can't smack the cells too hard either, you'll kill 'em. As a result, personally I use PI or 7ADD any time I take an adherent line through the flow so that I gate out the dead cells. Assessed with PI exclusion, I've had an all time high of 92% viable, and as little as 42% (ouch!)... Even with gating, the data wasn't easy to interpret at 42% viability either. Hopefully, you have lots of the cells you wish to use so you can determine what conditions you need to use to 1) maintian viability, and 2) get as many single cells as possible. Hope this helps, David ============================= David L. Haviland, Ph.D. Asst. Prof. Immunology University of Texas - Houston, H.S.C. Institute of Molecular Medicine 2121 W. Holcombe Blvd. Houston, TX 77030 Internet:"dhavilan@imm2.imm.uth.tmc.edu" http://www.uth.tmc.edu/~dhavilan Voice: 713.500.2413 FAX: 713.500.2424 "A conclusion is simply the place where you got tired of thinking." =============================
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:50:21 EST