Hi Mariusz, I have had the great pleasure of having to sort keratinocytes in one form or another for the past 12 years or so, so I can assure you that it is possible. However there are a number of things to be borne in mind. The sample preparation is important; keratinocytes have this unfortunate tendency to stick together (although I think skin wouldnt quite be the same if they didnt), so the first thing is to try to get as close as possible to a single cell suspension. Trypsin, collagenase, EDTA in various combinations depending on the cell type and origin. Even after preparation, they can stick so EDTA or a bit of DNase in the medium will help, as will keeping them cool. Filtration prior to running will also help but you may lose a lot of cells that way. The worst samples are those that contain terminally differentiated keratinocytes (the suprabasal cells that are likely to be in abundance if you are using primary skin for example or differentiated cultures). These tend not to round up and can be a problem. If you are just using a benchtop anlayser though, with the right precautions, there shouldnt be too much clogging. Sorting brings its own problems as generally the orifice is much smaller and more prone to clogs. It is possible to use a larger nozzle - say 100um - but I find that a 70um is OK if the sample preparation is good (there is the possibility of fuzzy side streams with larger cells). It is best not to have the cells too concentrated and to run them at a relatively slow speed (I generally dont go over about 3000/second). the trick is to get them to run at a reasonable speed with as low a sample pressure as possible but without them being too concentrated. Be wary of having the sample tubing too close to the bottom of the tube and re-suspend the sample regularly. Again having the tube sheathed in ice is a good idea. Hope this helps and good luck! Derek On Thu, 24 Aug 2000, Mariusz Olejniczak wrote: > We are going to do some experiments with keratinocytes. Unfortuatelly > until now when we have traied to acquisite these cells it colged our > cytometer. Did you have the same expirence. How we could stop this problem ? > Mayby sombody have a protocols for flow keratinocytes investigation by flow ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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