Cytometry and Antibody Technology

Optimized Voltages on Benchtop Analyzers

by | Oct 18, 2019 | Learn: Basics of Flow, Learn: Cytometer Setup | 4 comments

The voltages on a flow cytometer are one of the more challenging settings to adjust properly. If voltages are too high or too low, the data looks terrible and is potentially unusable. Once voltages have been set and data have been collected there is no way to go back and fix it; the hideous data will just haunt you for the rest of your days. On top of that, the tips newcomers get on how to set voltages range from the outdated “Stick your negative population in the first decade of the axis”, to the vague “Keep everything on scale”, to the incredibly confusing “Adjust your voltage to minimize the compensation values”.
Does it need to be so complicated? Not really. In an effort to make this component easier, our facility now has a default experiment on each of the Fortessas with a suggested starting voltage for each of the detectors.

 

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”.

4 Comments

  1. I am working on immunophenotyping human whole blood samples.
    with three panels include 10 Antibodies in each panel.

    As I am new to this field and I could not adjust any voltage setting (Just move everything towards the negative site using just an unstained tube, not adjust any voltage using a single color tube)or I couldn’t set any staining titration of antibodies.
    Now after running several experiments I came across that different acquisition setting (Currently, I am using all 7 antibodies in each panel as 5 uL which suggested in datasheets from the companies),
    I m looking for different spillovers in the unusual channels, and as the acquisition (Voltage/gain) setting changing, I am looking for different spillovers in different channels.

    I need your help and guidance it will really help me to resolve the issue and I could optimize a good
    the experiment setup.

    1. To find out antibody concentration or to optimize voltage? which one I have to perform first and instead of whole blood if I could adjust antibody amount and voltage with the beads is it correct?
    2. To set the correct voltage, can I resolve the current problem which I came to know that spilling over in different experiments in different channels in single controls with different acquisition settings.
    3. After this all I will go for compensation set up do I am correct?
    4. Is it necessary to put Channels on which I am not using during experiments. for example, there is nothing labeled with ECD or V660 than is it necessary to put these channels on during my 10 color experiments.
    5. Can I optimize voltage with the same fluorophore but different antibodies only once. for example, CD95 PE and CD57 PE (they are not in one panel), if I optimized my volage for Only one antibody, can I used the same for another one as well.

    Best Regards,

    Suchita S. Jadhav

    (Ph.D. Student)

    Prof. Michael Firer reserach group,

    Laboratory for Immunology & Cancer Biology,
    Dept. Chemical engineering, Ariel university
    Ariel, 40700, Israel

    Reply
    • Hi Suchita,

      You may find this video useful: https://youtu.be/wTUviuik6rw. To answer your questions:
      1. Voltage is dependent on the cytometer, titration is dependent on the antibody. Voltage and antibody titration are not dependent on each other. The optimal voltage should be determined first and does not need to be done every experiment. You may find it easier to use beads to do a voltration test.
      2. I’m sorry, I don’t understand the question.
      3. Yes, compensation can only be done after voltages are set.
      4. You can remove channels you aren’t using if you want to.
      5. See my answer to #1.

      Reply
  2. Hi Laura,

    I was wondering, can I stain some cells for all the markers in my panel and run the sample at different voltages or it’s important to run individual (single stain) samples to complete voltration?

    Reply
    • Hi Elena,

      The voltration experiment is completely independent of a scientific experiment. A voltration experiment should be done once to determine optimal voltages for a specific cytometer. When running a scientific experiment, you can start with the optimized voltages and make minor modifications before running all of your controls and samples on the same exact voltage settings. For compensation to be calculated correctly the controls and samples must be run at the same voltages.

      Reply

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