At 05:41 7/10/99, Jeff_Carrell@hgsi.com wrote: >Does anyone fill their cell sorter sheath fluid tank "out in the open", or does >everyone fill the tank in the laminar flow cabinet? I have 3 tanks used in rotation, the cycle for any tank goes like this:- First the tank is emptied, rinsed and charged with fresh saline. A new 0.45um filter membrane is fitted, all openings are covered with alfoil and the whole thing is autoclaved. On a day when a sterile sort is scheduled, a fresh tank is used; If no sterile sort, then yesterday's tank is refilled "out in the open", but I will generally change tanks weekly even if no sorting. Each morning, the sorter lines are flushed with 70% ethanol for ten minutes, then a sterile 0.22um syringe filter is fitted on top of the normal filter holder and the saline flows through both filters. This pattern has worked very well for us for about 4 years now, I very rarely get a hint of a contaminated sort. There are of course many little details left out of the above description, feel free to ask... ------------------------------------------------ One aside: We do occasional calcium flux experiments, and on one particular day everything was positive, including non-stimulated negative control cells. That happened to be the day I was in a big hurry and left out the (daily) ethanol flush step. ;~( We stopped, did the ethanol flush and re-started, then the rest of the experiment went fine! There was apparently *something* in the machine giving the cells that calcium kick, and it was killed by ethanol.... ------------------------------------------------ After nearly 20 years I still have much to learn ;~) Joseph. -- Joseph Webster Flow Cytometry Facility Centenary Institute
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