Mark, Several points . . . 1) What's the viability like at start? Are you using a viability exclusion dye as part of your sort logic? You may find that the cells are in rough shape at the outset. 2) Are you sure these cells hold better at 4C? Often, human cells hold better at room temp, where rodent cells survive better on ice . . . something to consider. If you do, however, find that these cells survive better chilled, it makes no sense to cool the sort sample and not the sort collection . . . 3) Post sort viability has never been an issue here, but plating efficiency for one user has improved since I switched my sheath buffer (thanks, Rochelle Diamond, at Cal Tech) to HEPES (5mM, ph 7.4). The point here is that there may be something about your sheath buffer that's causing problems. 4) Clumps are a separate issue, but perhaps a little serum (0.5-2%), or some BSA (0.5%) in the original suspension will help. You might also see an advantage to adding a little DNAse (to eliminate clumping caused by extracellular DNA). Also, I never use the in-line filter, since (as you know) they clog rather quickly . . . hasn't been a problem for me. You might try running without it. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry <http://www.cancer.med.umich.edu/flow_cytometry> phone: 734-647-3216 fax: 734-936-7376 kukuru@umich.edu "Miller, Mark A." wrote: > 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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