CHOOSE THE OPTIONS THAT APPLY TO YOUR EXPERIMENT - This methods file was meant to be edited for your specific experiment so includes ALL options. You need to document which filter setup you used, etc for your image collection from these options.   METHODS AND MATERIALS FLOW CYTOMETRY  

Physiology and Biophysics Facility

Flow cytometric analysis was performed using the Becton Dickinson FACScan fluorescence analysis system equipped with a FACStation, MAC PowerPC 7600/200 computer and CellQuest acquisition and analysis software (for immunophenotyping) or CellQuest acquisition and Modfit LT DNA Analysis Software (for DNA analysis). This instrument is equipped with a 35mw argon ion air cooled laser at 488nm excitation, and data is collected using up to 4 photomultiplier tubes and one photodiode, 5 parameters and up to 1024 channels. Filter configuration includes DF530 /30 barrier filter for green (FL1) emission, DF585 / 42 barrier filter with a LP640 Dichroic filter to separate red (FL2) emission from orange (FL3) emission, and a LP 650 for FL3. Flow rates were set at HIGH ( 60 ul per minute) or Low (12 ul per minute). For optimum coefficient of variation (CV), lower flow rates were used (particularly for DNA analysis). Analysis was done on a collection of 10,000 (usually) events within a doublet discrimination gate (for DNA) or population gate (for immunophenotyping), or ungated. The doublet discrimination gate is drawn on a dot plot with the width of the signal on the X axis and the area under the peak of the signal on the Y axix, eliminating those which are wider - or multiple cells clumped together. The cells which fall in a somewhat diagonal line are singlets and those which fall to the right of that are clumped.