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Methods for optimizing PMT voltages on BD digital instruments

Protocol

Part I: Determining minimal application-specific optimal voltages

Objective: Determine a minimal desirable gain using the dimmest objects to be measured (usually unstained cells) where electronic noise does not make a significant contribution to the measured signal.

A reasonable minimal gain is one in which the variance of electronic noise (en) is not more than 20% of the total variance. To set the voltage (also known as gain) where the variance of electronic noise (en) is not more than 20% of the total variance we use a target of between 10% and 20% for unstained cells (i.e. 15%).

Since variance = SD2, we therefore want to set the gain where
SD2en=(0.15)*(SD2target)

Therefore SD target = equation image
Note: rSDen = robust standard deviation of electronic noise (this metric excludes the skewing effect of outliers)

The following three steps were performed to derive the rSD targets and the calculated values are posted beside each instrument

  1. Obtained rSDen from Baseline Report for each PMT.
  2. Calculated target rSD for each PMT (rSDtarget = equation image).
  3. Calculated upper and lower limits: rSDupper = equation image and rSDlower = equation image

The protocol below will guide you through a procedure in which you will adjust the gain until the rSD of your unstained cells equals the rSDtarget.

  1. Using the rSD target values from the posted chart, run unstained cells and adjust voltage until the rSD of the unstained peak roughly meets the rSD target and is within the rSD lower and rSD upper limits. If you have more than one cell type in your experiment, use the ones that you expect to have the lowest levels of autofluorescence for this step.
  2. Check dynamic range.

    Put on a fully stained sample at (minimal) target gain. Choose the samples that you expect to have the brightest total signal (i.e., autofluorescence plus fluorochrome signal) for this step.

    1. Ensure the positives are on scale in the linear range of the detector and not greater than ≈ 100,000 - 120,000. If they are at or above 200,000, lower the voltage until positives are ≈ 100,000 - 120,000. This will leave some "wiggle room" at the top end of the scale in case some samples are even brighter.
    2. Run compensation samples to ensure that they are also within the linear range of the detector (may exceed 100,000 but not into a nonlinear region) and staining is greater than or equal to staining levels of cells. If it's not there isn't anything that you can do at this point!
      Note: The linear range for each detector can be found on the baseline report.

Part 2: Capturing bead target values for future experiments

Objective: To capture bead target values for use in future experiments. This will allow you to set the instrument in a consistent manner from experiment to experiment.

  1. Create a worksheet template with histograms plots for each parameter.
  2. Run SPHERO Rainbow Calibration particles (Spherotech, cat# RCP-30-5A-4) to capture bead target values (i.e. the MFI of the beads for each channel). These are beads that contain a mixture of fluorophores that fluoresce in all channels.
  3. Create a gate around the peak on each histogram and note MFIs. You can export statistics as a CSV file that can be viewed in or printed out from Excel. Save worksheet template for use in subsequent experiments.
  4. Export and save the cytometer instrument settings by right-clicking the cytometer settings icon in the browser and choose "Export".
  5. For subsequent experiments, right-click an open experiment in the browser and choose "Import Cytometer Settings". Click "Yes" to overwrite the current settings. Select the settings file you want to import and click "Import".
    Note: Cytometer settings include PMT voltages, compensation, threshold and ratio values.
  6. Run beads and adjust voltages so that they meet the bead target values established in step 3.
  7. Proceed with experiment

    For cells whose autofluorescence will change from experiment to experiment (e.g. tumour cells, cultured cells etc.):

    Using the Lymphocyte Bead Target Values, highly autofluorescent cells will often sit in the second decade or higher in the Pacific Blue, FITC, PE or PE-TR channels. The amount of autofluorescence is much lower in the red and far-red channels. It is recommended that you DO NOT arbitrarily lower the PMT voltages to bring the "background" down to the first or second decade. This autofluorescence is real fluorescence and should be recorded as such as long as your stained cells are well on-scale. However, if your stained cells are off-scale on the upper end, then lower the voltages just enough to bring their MFI to 100,000-200,000. After that, capture bead target values for future experiments as described above.