When loading your tube into the FACS machine, run the unstained / IgG control sample first as this aids in setting the baseline parameters
Check your event rate in the Acquisition Dashboard window. If it is greater than 1500 evt/s turn down the “flow rate” or unload and dilute the sample further. If less than 100 evt/s, turn up the “flow rate” (don’t exceed a flow rate of 5.0 if possible, as the instrument is less focused and more inaccurate at higher flow rates)
In the Acquisition Dashboard window choose the appropriate “stopping gate” and “storage gate” (when working with nuclei, set as “Nuclei” and “All events” respectively)
Choose the range of “events to record” and “events to display” that best suits your purpose (≥ 5000 for both is advised)
Under the “threshold” tab in the Cytometer window, change the threshold (should be set for FSC) so that any small events in the bottom corner of the FSC vs SSC graph (caused by general cell debris and dust) are no longer shown. The threshold should not be set too high so that it causes an arbitrary, artificial cut-off through the left side of your population but not so low that small events caused by debris/dust are visible (ideally between a threshold 200-500).
Under the “parameters” tab in the Cytometer window, adjust the “FSC” and “SSC” values to get your population sitting in the centre of the FSC vs SSC graph (a re-adjustment of the “threshold” may be required at this point). It is essential to select “restart” each time any of the parameters are changed to update the events being displayed to ensure only events are recorded under the new settings.
Adjust or draw a new gate in the FSC vs SSC plot to encompass the population of interest.
Look in the scatter graph of SSC-A vs SSC-W (if you opened a blank experiment you will need to draw one). Right-click on the graph and check it is only displaying the events encompassed by your previous FSC vs SSC gate. Adjust or draw a gate for SSC A vs SSC W to encompass all of the main population to the left of the graph and exclude outliers to the right (these are doublets and other cell debris clumps)
Under the “parameters” tab in the Cytometer window adjust parameters for the fluorochromes selected so the unstained / IgG control sample sits close to 0 for the fluorochrome on a graph of FSC vs fluorochrome.
Load the stained samples and check the stained population has a clear increase in signal for the fluorochrome in comparison to the unstained (signal should not exceed 10x4). Several minor re-adjustments of the fluorochrome’s “parameters" may be necessary for the stained sample at this stage. If so, the unstained / IgG control has to be reset and re-recorded.
12. Regularly check your “Efficiency” in the Sort Layout window value. Between 80-100% is ideal, 70% is acceptable if less than 70% either the sample is too concentrated or you are sorting a rare population. Although the “flow rate” in the Acquisition Dashboard window can be increased to make the sort quicker, faster flow rates are less efficient.
13. Check the “Electronic abort rate” (N° errors /sec) and “Electronic abort count” (Tot N° of errors) at the bottom of the Acquisition Dashboard window. These parameters measure potential miss-sorts (different from efficiency as efficiency measures undetermined drops which are directed to the “Waste” and therefore lost but do not contaminate).“Electronic abort rate” should be <1% of total events per second.
14. For long sorts, gate positions should be regularly monitored, especially for stained populations as fluorochromes lose intensity over time and the population can shift towards the unstained. Gates can be moved during long sorts to compensate.