While many fluorescence-based flow cytometry assays can be run without concern for hitting the exact same fluorescence intensity target values across different experiments, there are assays that require standardization such that assays run on different days, or even different flow cytometers will render results that can be directly compared. BD Biosciences has developed a protocol for enabling “Application Settings” on machines running the FACSDiva V6 software, to create settings that allow for standardized consistent fluorescence intensity target values to be obtained across experiments run on different days or instruments.
The ability to collect replicable fluorescence intensity values across assays is invaluable when assays require directly comparable results. This can easily be envisioned for use in the clinical setting where specifically defined expression levels of a given marker may be used for prognostic or diagnostic indications or therapeutic responses. Other examples include assays where the number of samples is too large for an experiment to be logically performed on a single day, or time series experiments where samples will be assayed for the same parameters and assessed for their changes over time.
Creating an optimized Application Settings for a given antibody staining panel requires several steps and the understanding of several principles of flow cytometry, and a thorough reading of the protocol referenced at the end of the article is recommended. Here I will discuss the basics for generating Application Settings and using them on the same cytometer. For a more detailed explanation of the procedure and how to apply this to additional instruments which have the exact same laser and detector configurations, please refer to BD’s protocol.
In the first step of this protocol, the user runs the standard Cytometer Setup and Tracking (CS&T) software using the CS&T beads. This will determine the optimized photomultiplier (PMT) detector voltages for the CS&T beads to minimize electronic noise while maintaining the brightest fluorescence staining in the linear range of the detector. However, these voltages are optimized for these beads and not for cells. Thus, once the CS&T report is generated, the next step is to optimize the settings for the cells and stains of interest.
In the next step of this protocol, the objective is to adjust the voltage settings to optimize the balance between the electronic noise and the linear range for each detector using the same cells and fluorescent antibody stains for which the application settings are being created. The negatively stained cell populations are desired to be located above the noise on the low end, and the positively stained cell populations need to be within the linear range of the measurement.
The electronic noise robust standard deviation (rSDEN) is determined by the CS&T software and indicated in the CS&T report for each detector. To determine the optimal location between the noise and background for the negatively stained cell populations, a calculation of 2.5 x rSDEN is made for each detector. Then the voltages are adjusted while running the negatively stained cells to place them at this median fluorescence intensity (MFI) value for every detector being used.
After adjusting for all of the negative populations, the positively stained cells must be assessed to ensure that the cells are still within the detector’s linear range. The CS&T report also generates a linearity max channel value for each detector. Thus, the MFI of the positively stained population should be below this value, and allow for any anticipated increases in expression to remain below this value. Remaining within the linear range is more important than having the negative populations at the 2.5 x rSDEN levels. Thus, when there is very bright staining, this is important to keep in mind and the voltage should be lowered accordingly.
Finally, after the user has adjusted the voltages to the optimal settings for each detector, the application settings can be saved using the FACSDiva V6 software under: Cytometer Settings -> Application Settings -> Save.
Once the application settings have been created, they can be applied in any future experiments. To use them, following daily CS&T cytometer setup and performance checks, open a new experiment. Apply the Use CS&T Settings selection. Then open the application settings under: Cytometer Settings -> Application Settings -> Apply. Now consistent MFIs should be obtained for cells run on different experimental days. One final note is that additional considerations need to be taken if the baseline target values change over time, or a new lot of CS&T beads is used for the daily cytometer setup. The BD protocol discusses what to do in these instances.
Standardizing Application Setup Across Multiple Flow Cytometers Using BD FACSDiva™ Version 6 Software. Ellen Meinelt, Mervi Reunanen, Mark Edinger, Maria Jaimes, Alan Stall, Dennis Sasaki, Joe Trotter.