Moxi V
™
User Guide
Page 25
the green representing the fluorescence- (below the red line) points. Any areas of overlap in the
histogram (between red and green populations) are shaded yellow.
Note: The overlap is a
natural consequence of converting the 2D scatter plot data to a 1D Histogram.
The two black
boxes below the histogram provide the subset concentration (cells/ml), mean particle diameter
(
Note: This is an “effective” diameter that is based on a precise volumetric measurement
(Coulter Principle) combined with a spherical assumption.)
, particle volume (directly measured
by the Coulter Principle approach), and median fluorescence intensity (MFI) values for the
negative (green, “Lower Population”) region and the positive (red, “Upper Population”) region.
size-only tests, the size-region is determined by positioning of blue gate markers directly on the
histogram (as described in the “Size Histogram” Assay section above) and the histogram is only
shaded green. For size histograms displayed for fluorescence tests, the size-gated region of
the primary (display channel) fluorescence vs. size scatter plot is used to determine which
points are included in the histogram binning.
Simplified Output and Auto-Gating Overview
The two main outputs listed above are the main way raw data is viewed and analyzed.
Ultimately, the data needs to be “gated” to properly identify sub-populations by size and
fluorescence intensities. The system has a couple of built-in algorithm for “auto-gating”
populations. For size-only (no fluorescence) outputs, the system offers a curve-fitting algorithm
that automatically identifies a core Gaussian cell population, allowing for automated
gating/counts of monodisperse (single cell type) samples.
The system can also auto-gate the Fluorescence vs size display to identify both cell populations
(i.e. size gating) and fluorescence positive and negative (e.g. live/dead) populations. For the
Cell QC, CAR-T, and PBMC assay, this algorithm is automatically implemented to provide a
simplified, quick-read output to end-users. Even with system auto-gating or curve-fitting, users
always have the ability to view the gated histograms and scatter plot to critically analyze the
gates and adjust them as needed.
All data outputs are discussed in detail below first for test-specific and simplified outputs
followed by general gating instructions (for the three main data formats) in the “Manual Gating”
section. The general gating instructions are applicable to all assays.
