How did we determine starting voltages?
Each detector on each instrument has a “sweet spot” or optimal setting. In order to determine these optimal settings, we ran the same tube stained with one antibody at 6 different voltages (Figure 1). This voltration test clearly shows what happens when the voltage changes: at lower voltages it may be difficult to resolve the positive population from the negative population, but at higher voltages the negative population begins to spread out and/or the positive population is off the plot (saturating the detector). We can assess these different voltages numerically by calculating the separation index or staining index (SI). The SI takes into account the distance between the means of the positive and negative populations as well as the spread of the negative population. Ideally, we want to use a voltage where we have the highest SI. Figure 2 demonstrates that increasing the voltage does increase the SI – to a point. Eventually, the SI will plateau and increasing the voltage will not improve the data.
So how did the CAT Facility choose the optimal starting voltages? First we stained mouse spleenocytes with a single fluorophore. Using the same clone of anti-CD4 and the same concentration of antibody (0.1 ug/tube) we created single stained samples for most of our frequently used fluorophores. After running each tube at 6 different voltages, we calculated the separation index and selected the optimal voltage for each detector.
For other fluorophores that we did not run ourselves, we used an alternate method. BD instruments have automated software called Cytometer Setup & Tracking (CS&T) that is used to characterize, setup and track the instrument performance. One part of CS&T is determining an optimal voltage for each detector. So why did we do our own voltration test instead of just using the CS&T? CS&T beads are used to determine optimal voltages – these beads contain an unstained, dim positive and bright positive bead. The optimal voltage is then calculated by determining the highest SI for separating the dim positive bead from the negative bead. The problem with that is fluorophores that are dim require a very high (above 700) voltage to resolve the dim positive bead from the negative bead. For best results, panels should be designed so that dim fluorophores are paired with highly expressed markers. If a panel is designed properly, there should not be a need to resolve a dim population in a dim fluorophore. When comparing our voltration experiment results to the CS&T results, we had similar voltages for most flurorophores. However, for dim fluorophores, we chose to instead use a voltage that optimally separated the bright positive population from the negative population.
How do you use the optimized voltages?
We suggest starting with the optimized voltages in the default new experiment. If the single stained controls are so bright that they are no longer on the plot (cells are on the right Y-axis), then the voltage should be lowered for that detector until all of the positive cells are within the plot. Increasing the voltage above the recommended voltages is not beneficial, as shown in Figure 1.
Where can you find our data?
All of our results are posted on the resources page under “Benchtop Analyzer Voltages and Staining Indexes”.