GeminiBio MXG102, Руководство пользователя

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Moxi GO II
™
User Guide
Page 61
on the PMT2 scale as the dark cluster). The third row shows an example of compensation for a
dual-label (FITC-Annexin V and Propidium Iodide) Jurkat Apoptosis test. Note that the applied
compensation only affects the cluster in the lower/right, which corresponds the FITC-
A/PI- population. For Annexin/PI assays, by nature, whenever a cell is PI+
(membrane is permeable), it is also Annexin V+. So this population does exhibit a slight upward
slant due to the correlation of Annexin and PI staining (and due to DNA content differences as
well). However, it does not exhibit the same “artificial” narrowness of the spillover populations.
Compensation with Negative Control (Load Control Set)
For many single-stain positive controls, there is no fluorescence-negative (dark) population to
use to level the spillover cluster with compensation. In those cases, the user needs to first run a
negative control test. That test can subsequently be used to compensate the single stain
positive control sample by using the on-board “Load Control Set” compensation functionality.
The workflow for that functionality is shown below. Looking at the image below, the screenshot
at the top/left picks up from the point when the single-stain control test has completed acquiring
data, and the user touches the “Comp” button to begin the compensation process. In this case,
you can see that there is no “dark” population (i.e. all the beads are fluorescent). And, you can
see the characteristic tight-slant of that population, an indication of the spillover effect. In this
case, the user can touch the “Load Control Set” button to select a previously-run negative
control data set to use. The system will display the full “Saved Data” list (image below –
top/middle) for the user to touch/select that set. Once selected, the system will load that data
and superimpose it (in gray) on the single-stain positive control scatter plot (see image below –
top/right). As before (see instructions above), the spillover channel (in most cases PMT2)
should be displayed on the y-axis. If it is not, the user can touch “Switch Axis”. At this point, the
compensation can proceed as described above. The user needs to adjust the gates by
touch/dragging the red and blue dots to get the spillover cluster in the bottom/right quadrant and
the control cluster in the bottom/left quadrant (as shown in the image below – bottom/right
screenshot). The yellow box at this point shows the MFI (mv) difference between the single-
stain positive control (points in the bottom/right quadrant) and the control cluster (points in the
bottom/left cluster). At this point, the user can touch “Auto Adjust” to have the system
automatically calculate an applied compensation level. The user can also use the up/down
arrows to manually adjust or fine-tune the system calculated compensation level.
Note: The
single arrows adjust the compensation level by 0.1% and the double-arrows by 1.0%. Rapidly
tapping the arrows will register multiple increment changes PRIOR to refreshing the screen.
When complete, the spillover cluster should be positioned at the same height (on the y-axis) as
the control cluster and the delta MFI should be close to zero (see image below – bottom/left, try
to minimize value to an absolute value <5mV).