Dear Mark, I manage a facility with a MoFlo high speed cell sorting cytometer. I have been involved in sorting bacteria much smaller than E.coli. I get excellent results using a 100 um tip & about 50kHz drop gen. frequency. I agree that event rates above 25,000/sec a 75 um tip & 100kHz drop gen. would be better. The problem as I understand it is the speed at which the software runs. 180,000 decisions(cells)/sec is fine in theory, but a lot of software (I believe most) cannot cope. The size of the drop is essentially unimportant, its how many cells you get inside the drop that makes a sort good or bad. This is your determination on purity. For a library I assume you are looking for 1 pos. cell/drop, with no other cells (pos. or neg.) present. This is not always best fixed by sorting faster. High speed sorting usually means a high coincidence rate = more pos. cells discarded. How you create your library also determines the rate at which you sort. I can sort directly into a 96 well plate, but the ultimate speed of the sort is dependant on how fast the robotics can move the plate to catch the drop. Mark, no one who wishes to learn is ignorant, only less informed, & nobody knows everything. I too am new to flow cytometry, its whole different game to routine histology & electron microscopy. Regards Rob W. At 12:54 AM 2/26/99 -0500, you wrote: >I am a chemistry grad student, and my project is to sort genetically >engineered libraries of E. coli. I am currently investigating ways to >increase the rate of sorting on a commercial droplet sorter. Most of the >commercial machines have a 76um orifice, which is much bigger than coli or >yeast. I have noticed a reference from Shapiro's Practical Flow Cytometry >(3rd) by Fellner-Feldegg on using a small 25um orifice. Is it realistic to >fit a BD Vantage or a Coulter Altra with say a 20um orifice and a 100kHz >droplet generation frequency, and thereby increase the effective sort rate? >I am guessing that a cycle time of 5.5 microseconds means a maximum of >180,000 decisions/sec. This seems acceptable? I noticed that the >Cytomation MoFlow uses droplet deflection technology, but would the BD or >Coulter drop deflection plates result in acceptable purity and yield? Is >jet-in-air required for these kinds of sort rates? Are there other >considerations that need to be taken into account? I apologize for the >ignorance, and thanks in advance. R. Wadley, B.App.Sc, M.L.S Laboratory Manager Cellular Analysis Facility School of Microbiology & Immunology UNSW, New South Wales, Australia, 2052 Ph (BH) +61 (2) 9385 3517 Ph (AH) +61 (2) 9555 1239 Fax +61 (2) 9385 1591 E-mail r.wadley@unsw.edu.au www http://www.unsw.edu.au/clients/microbiology/CAF.html (Under development)
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