What you need to know as a User:
When you launch FACS Diva and you are recommended to click “Use CST settings”. The reasons are that :
- When a check performance is run (performed routinely by the Core staff), the PMT voltages are adjusted to keep MFI values the same. This is done by the software (i.e not the operator) and is done so the fluorescent quality control beads meet the standards set by BD.
- Optimal gain (i.e voltage) can reduce errors having to do with resolution and identification of dim populations
- Over time, lasers (especially the red laser) will experience vertical drift. This is why laser re-alignment occurs every 6 months, or sooner if needed. Time delays* are set with CST to accommodate laser movement to avoid loss of signal. Signals are aligned with respect to the blue laser, so the red laser signals always have a negative delay value. Time delays can also be affected by sheath air pressure or the sheath tank going from full to empty.
- Window extension* is also determined by the CST.
Time delay: Most multiple laser sources are spatially separated and create emission pulses from a single cell (event) at different time points. The Time Delay Calibration is used to align these events in time and must be accurate to within a fraction of a microsecond for inter-laser compensation to be accurate. We do not change this.
Window Exstension (WE) is time interval during which the input signal is greater than the threshold and during which the acquisition system looks to sample a pulse. We do not recommend changing this and the software automatically defaults to an instrument-specific WE. Please see the following for a more detailed explanation: An Introduction to Window Extension on Digital Flow Cytometers.
Additional benefits to using the CST software?
BD CST allows the generation of application settings using BD CST bright bead target values for each fluorescence detector to obtain consistent and reproducible results over time. These settings are linked to the CST Settings and if one parameter changes the application settings adjust the voltages for the parameter. The result is, that the populations remain on the same position at the plot.
This can be of use when doing kinetic studies, or cGMP/cGLP studies.
What can it not provide?
Optimal design of your experiments. If you have “strange populations” or effects during the measurements please come to the Core staff and discuss the problems with us. We will assist you in troubleshooting the problem.
We run quality control beads routinely to aid in troubleshooting the instruments. FACS DIVA software (and later) has an in-built QA software program call Cytometry Setup and Tracking which we use to evaluate the instruments. We have similar Quality Control problems for the ImageStream and the Attune NxT.
Note we do not allow users to run baseline CST, change the optical filters or laser configuration templates. Please see the Core staff if this needs to be done for your experiment.
A Quick Summary of What the Core Staff Do:
1. Every 6 months: Run a baseline after maintenance (BD service contract) is done on the instruments. The baseline sets the laser delay, it evaluates linearity, detector efficiency, optical background and electronic noise. PMT voltages are based on what will give the best resolution in each detector determined by the above factors.
2. Daily for Analyzers: Run a daily performance check on the analyzers to verify that the cytometer is performing within its constraints. PMT voltage, area scaling and laser delays are adjusted by the software. It’s very important to accept the CST settings so that you do not lose signals due to laser movement and have optimal resolution.
3. Prior to Sorting: A performance check is run through CST. The drop delay is then calculated using BD FACS Accudrop beads. Determining drop delay (either manually or automatically) is required to properly sort cells.
4. Post-sorting: Sorter and lines are cleaned and decontaminated to prevent contamination of upcoming sorts. For detailed cleaning protocols, please see: Decontamination of the BD FACSAria II or BD FACSAria III System Using the Prepare for Aseptic Sort Procedure.
5. Weekly: The Core staff prepare on average 40 to 60 litres of PBS per week. Sheath fluid is a required for running the machines. Occasionally special/customized sheath fluid is prepared. Please discuss this with the Core staff if this is required. For sorting we prepare the 10L sheath fluid as fresh as possible and autoclave the tanks on a monthly basis.