It is a truism that a clean sorter is a happy sorter. It is important to make sure that all fluidics lines be cleaned and/or replaced on a regular basis. If a sort is aseptic (i.e., one in which cells will be required to be recultured or transplanted), a sterilization procedure will be needed. In general, this involves running 70% ethanol through all fluidics lines for 30-60 min before flushing this with distilled water (30 min) and finally sterile sheath fluid (at least 30 min before commencing a sort). At all times, fluids pass through a 0.22-|im filter immediately after leaving the sheath tank. It is important to remember that the compressed air used to generate the pressures needed to run a sorter should also pass through an in-line filter and that all filters should be replaced periodically. All areas where the cells can potentially be in contact with the atmosphere (i.e., the sample line and sort chamber) should also be cleaned with ethanol prior to a sort. To check the sterility of a flow sorter, it is useful to periodically remove fluid from key locations (sheath tank, nozzle, and sample line) and put this into culture in an appropriate medium; cultures should remain sterile for at least 7 d.
The next consideration is nozzle size. The cell type will influence the size of orifice used. A general rule of thumb is that for blockage-free sorting and coherent side streams a cell should be no more than one fifth the diameter of the nozzle. In practice, this means that small round cells such as lymphocytes or thymocytes would require a 70-^m nozzle whereas many cultured adherent cell lines and primary cells such as keratinocytes would require a 100-|im nozzle. Other larger cells such as plant cells require larger nozzles, typically approx 150-200 ¡im (45). In practical terms, a larger nozzle will use more fluid and will need a longer time from the emergence of the stream into air to break up into drops, because it will run at a lower pressure.
In contrast to analytical benchtop cytometers, laser alignment should be checked on a daily basis on a stream-in-air system. It is important that the laser or lasers hit the stream in parallel and that the beams be focused correctly. Alignment may be checked using fluorescent latex microspheres (beads) that are excited by a particular wavelength of light and have a broad emission spectrum; it is also important to monitor the sensitivity of the sorter using multipeak beads with a variety of fluorescence intensities. The number of lasers used and the fluorochromes to be detected will also vary and need to be determined before the flow sorter is prepared.
Once aligned, the frequency and amplitude of the drop drive are altered to achieve a minimum stable break-off with coherent side streams using a test-mode sort. Although a given nozzle size has an approximate resonance frequency (Table 1), there will be a variability between individual nozzles of a given size and there will also be variability on a day-to-day basis depending on conditions.
A stable break-off point will be found at several harmonic resonance frequencies and only by experience will an operator be able to find the most stable of these. It is possible to alter the amplitude of the transducer during the sort to keep drop break-off stable and keep the charging pulse in phase. Many modern sorters have automated systems that will detect movement of the break-off point and change the amplitude accordingly. If the change in the break-off is greater than user-defined parameters, the sort will interrupt and alert the operator. It is always worth rechecking drop delay if there is a block and/or the break-off seems unstable.
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