Dear Frederic, using fixed (paraformaldehyde or glutaraldehyde) cells, we sort with a 500x dilution of our sheath fluid, standard PBS, onto silane-coated or uncoated slides. This greatly reduces (but does not completely eliminate) formation of salt crystals. At this dilution, much of the salt in the drop comes from our isotonic sample buffer, as compared to the salt in the sheath fluid. This dilution is about the limit for stable side stream formation on our facstar+. You may have to adust the dilution for your instrument, and we find that we have to use a higher drop drive amplitude and twiddle a bit more with amplitude, stream deflection and phase adjustments than when using undiluted PBS. After drying, cells are readily visible in the microscope on a slight background of salt crystals. For photography, we rehydrate the drop with a few ul of distilled water and add a cover slip, this works quite well. You may have to mark the position of the sorted drop, with little salt buildup it can be difficult to locate after drying. It might also be possible to do this with initially viable cells, providing they can handle the relatively short period of hypotonic exposure before the puddle of sorted drops dries. >will those who sucessfully sort cells directly onto coated glass slides >for subsequent morphologic/cytochemical staining /assessment, kindly >suggest their preferred method to prevent salt crystals from forming? >kindly also respond with favorite technique and reagents for fixation. > >many thanks >f preffer >Frederic I. Preffer >Department of Pathology >Charlestown Navy Yard- 7140 >Massachusetts General Hospital East >Charlestown, MA 02129 > >voice [617] 726-7481 >fax [617] 724-3164 -- Jan Grawé Inst. för Genetisk och Cellulär Toxikologi Wallenberglaboratoriet Stockholms Universitet S-10691 Stockholm Sweden Tel +46-8-163666 mobil+46-70-5667874 fax +46-8-164315 epost jan.grawe@genetics.su.se http://www.genetics.su.se/gentox
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