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MACSQuant® Instrument quick guide
How to create an instrument setting
Setup of detectors
1.Perform PMT calibration (refer to the MACSQuant
Instrument quick guide ‘PMT calibration’ for details).
2.In a new workspace, open a template and acquire
some unstained cells
3.In the ‘Channels’ tab in the sidebar adjust voltage of
scatter and fluorescent detectors to set cell populations
on the desired position in the plot. Select a trigger
channel and set an appropriate value.
Note: Details on manual detector adjustment can
be reviewed in the MACSQuantify™ Software guide
Setup of automated compensation
Figure 1 : Rack configuration for CompensationMultiColor
1.Prepare single-stained controls representing all
fluorochromes to be used in the experimental staining
panel. Ensure that there is a comparable positive and
negative population for setting compensation.
2.Select the ‘Chill5’ rack from drop-down list in the
‘Experiment’ tab.
Note: It is recommended to have one tube that
represents a true blank.
3.Click on the ‘Rack’ button
in the toolbar.
4.In the ‘Racks’ dialog box, the Chill 5 Rack will be
displayed. Select the appropriate number of sample
positions to match the number of samples that will
be used for compensation.
5.While all wells are selected (orange), click on the
‘Group’ button at the bottom of the window. Each of
the selected rack positions should now be labeled with
the number ‘1’.
Figure 2: Settings for CompensationMultiColor and fluorochrome
selection
8.To set the fluorochrome for each rack position, highlight
the respective circle in the rack window. Only one circle
must be selected (orange highlight) at a time. In the
‘Sample ID’ field a drop-down list will be available from
which the representative fluorochrome can be chosen. 6.Choose ‘Express’ from the ‘Settings’ tab within the
‘Experiment’ tab.
9.Each fluorochrome that appears in the drop-down list
represents a specific detection channel. For example,
PerCP-Vio770™ represents the channel B3, therefore
using another B3-compatible fluorochrome, e.g,
PE-Cy™5, PerCP-Vio700™ should be selected.
7.Select ‘Setup’ from the ‘Type’ drop-down list and
‘CompensationMultiColor’ from the ‘Mode’
drop-down list.
10.The position ‘Blank’ is selected for a sample of
unstained cells. This is the reference for negative cells,
which should always be included.
Note: Sample should be arranged in columns, not rows.
11.The position ‘PI’ is selected, if propidium iodide (PI)
will be used for dead cell exclusion in later experiments.
It is used for compensating all fluorescence channels
against the channel B3. This ensures that there is no
fluorescence spillover into the B3 channel. PI itself
will not be compensated.
Note: For compensation use unstained cells and
do not add PI.
12.Place samples in proper rack positions and start the
acquisition. Then follow onscreen instructions.
Manual compensation using
compensation matrix
1.Choose an analysis template (e.g. four dot plots) by
clicking on the ‘New analysis window’ button
in the toolbar.
2.In the left dot plot, set up to visualize FSC on the x-axis
and SSC on the y-axis.
3.Change one dot plot into a histogram and display the
respective channel of the stained particles (e.g. for FITC
use B1).
4.In the ‘Experiment’ tab, program all necessary
parameters, such as Sample ID and Uptake volume.
5.Open the compensation matrix by clicking the
‘Instrument settings’ button
in the toolbar or
under ‘Edit > Instrument settings’ and selecting
the ‘Compensation’ tab.
6.Click the ‘Matrix’ checkbox.
7.Place the first single-stained tube (e.g. FITC-stained cells)
into the single tube holder.
8.Start acquisition.
9.When events start to appear on the plots pause the
measurement by right-clicking the stop button in the
instrument status bar and choosing ‘Pause’.
10.Draw a region around the population of interest (e.g.
lymphocytes) within the FSC versus SSC dot plot. This
will be P1.
11.Display only events in P1 in the FITC histogram plot,
by selecting ‘P1’ from the drop-down menu of the
plot header.
12.Draw two interval regions such that the positive and
negative populations are in two separate regions (e.g.
P3 for FITC positive and P2 for FITC negative
populations).
Figure 3: Setup of histograms and regions
13.Display the medians for all spillover channels used in the
experiment by clicking on the button, selecting the
spillover channels (e.g. B2, B3, etc.) and ‘median’ from
the ‘Feature functions’ tab.
14.Resume measurement by clicking on the ‘pause’ button
in the instrument status bar.
15.To add compensation to the combination of channels
in the plot, choose the appropriate cell in the matrix.
The columns represent the fluorochrome being
measured, the rows represent the detection channels,
where the spillover fluorescence should be corrected.
For example, to compensate a FITC-stained sample
against the PE channel, go to the cell ‘FITC/B2’ and
adjust the value to achieve equal median fluorescence
intensities for the positive and negative populations.
16.Increase the value if the median of positive population
is higher than the median of negative population.
Decrease the value if the median of positive population
is lower than the median of negative population.
17.Adjust values for other spillover channels, if necessary.
18.Once compensation is adjusted for this fluorochrome,
repeat compensation for all additional fluorochromes.
19.When finished, save as an instrument settings file.
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