I am having some trouble sorting HUH-7 human liver cells. The post sort viability is quite low, and I have to interrupt the sort every hour or so to remove the filter tips I am using because they are plugged up with cell clumps. I make the sample filters by welding 53 um nylon mesh to the large end of a 20 uL pipette tip. (I'm using a 100 um nozzle.) My collaborator needs to collect as many cells as possible each day we do this. The population we are looking for ranges from 0.5% to 5%. The cells are suspended at 1.5 to 2e6/ml in dPBS without Ca++ or Mg++. The sample tubes are kept on ice before they are mounted on the sorter and they are jacketed (4 C) while sorting. I am running a Vantage SE at 9 psi, 21 kHz, Normal-R with a 2 drop envelope. The cells are being sorted into 15 mL conicals with 10 mL of F7 medium supplemented with FBS. I do not have the collection tubes water jacketed. Any suggestions for decreasing clumping and/or improving post-sort viability would be greatly appreciated. Mark A. Miller Pharmacology Cell Sorting Lab WP27E-120 Mailbox WP26-265 Phone (215) 652 4597
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