GeminiBio MXG102, Руководство пользователя

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Moxi GO II
™
User Guide
Page 35
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.
Fluorescence vs. Fluorescence Scatter (Dot) Plot
Image above/right: Only the points within the size-gated region for the LAST VIEWED
fluorescence (PMT) channel vs. size scatter plot are included in the fluorescence vs.
fluorescence display. This is deliberately implemented so that just the cell (debris and noise
excluded) population can be analyzed. The scatter is visually divided into four quadrants by the
positioning of the gates. For each quadrant, a black box containing the quadrant statistics is
placed adjacent to the quadrant. Included in the box are particle concentration (cells/ml), MFI’s
– displayed as (x-axis MFI, y-axis MFI), and percentage of total counts (in yellow). The total
counts for all four quadrants is provided in the black box at the bottom of the scatter plot.
Simplified Output and Auto-Gating Overview
These three 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 any PMT 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.
The three core, two-channel cell health assays also have a simplified data output/representation
of the PMT vs PMT views that shows a Table and Chart view of the four quadrant data
(PMT1+/PMT2-, PMT1+/PMT2+, PMT1-/PMT2+, PMT1-,PMT2-).
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.