MACSQuant VYB Guide

MACSQuant VYB
Operator Training
LRI FACS lab
TABLE OF CONTENTS 1.
GENERAL OVERVIEW ......................................................................................................... 2
2.
OVERVIEW OF THE INSTRUMENT PARTS .............................................................................. 3
3.
...................................................................................................................................... 3
3.
3. OPTICS ........................................................................................................................ 4
4.
GETTING STARTED IN THE CUSTOM MODE........................................................................... 5
4.1
4.2
5.
5.1
5.2
5.3
CHECKING THE INSTRUMENT STATUS .......................................................................................... 5
DAILY AUTOMATIC CALIBRATION WITH MACSQUANT CALIBRATION BEADS ................................................ 7
QUICK GUIDE TO THE SOFTWARE MENUS ............................................................................ 9
TOP MENU BAR ICONS ........................................................................................................... 9
GENERAL MENUS ............................................................................................................... 10
TABS ............................................................................................................................ 13
6.
SETUP THE EXPERIMENT: ................................................................................................ 14
7.
MULTISAMPLE PROCESSING ............................................................................................. 17
7.1 SETUP A SAMPLE ON A RACK .................................................................................................. 18
7.2 SAMPLE GROUPING ............................................................................................................ 19
WHAT IS THE PURPOSE OF GROUPING SAMPLES? .................................................................................... 19
HOW TO GROUP SAMPLES? ............................................................................................................ 19
7.3 AUTOMATIC LABELLING OF SAMPLES ......................................................................................... 20
8.
8.1
8.2
8.3
9.
9.1
9.2
WORKING WITH REGIONS AND GATES .............................................................................. 23
HOW TO CREATE HIERARCHICAL GATES WHILE ACQUIRING MY SAMPLES? ................................................ 23
“NOT” GATES ................................................................................................................. 24
STOPPING GATE AND LIVE GATE .............................................................................................. 24
CHANGING THE PROPERTIES OF A PLOT, HISTOGRAM OR TEXT TABLE .................................. 26
OVERVIEW OF THE “PROPERTIES” SETTINGS FOR DOT PLOTS, DENSITY PLOTS AND HISTOGRAMS...................... 26
OVERVIEW OF THE “PROPERTIES” SETTINGS FOR THE STATISTIC OPTION. ............................................... 27
10. COMPENSATION ............................................................................................................. 28
10.1
10.2
10.3
AUTOMATED COMPENSATION USING THE EXPRESS PROGRAM COMPENSATIONMULTICOLORS ........................ 29
MANUAL COMPENSATION WHILE ACQUIRING USING THE FLUORESCENCE MEDIAN TECHNIQUE ........................ 31
RECOMPENSATION OFFLINE WITHIN MACSQUANTIFY SOFTWARE AFTER DATA ACQUISITION. ....................... 32
11. PRE-ENRICHMENT OF THE SAMPLE WITH MACSQUANT INSTRUMENT ..................................... 35
11.1
11.2
11.3
USE OF MACS TECHNOLOGY FOR PRE-ENRICHMENT WITH THE MACSQUANT COLUMN .............................. 35
PROPERTIES OF THE COLUMN ............................................................................................... 35
ENRICHMENT MODES ........................................................................................................ 36
12. OPENING AND SAVING DATA FILES, EXPERIMENTS AND INSTRUMENT SETTINGS ................... 37
13. EXPORT FILES, TEMPLATES, INSTRUMENT SETTINGS AND SAMPLE INFORMATION .................. 38
13.1
13.2
TRANSFERRING .FCS FILES, TEMPLATES AND INSTRUMENT SETTINGS USING THE COPY FUNCTION ................... 38
EXPORT SAMPLE INFORMATION AS AN EXCEL SPREADSHEET ........................................................... 39
14. TROUBLESHOOTING ........................................................................................................ 41
1. General Overview
The MACSQuant VYB is a benchtop flow cytometer that has been specifically
designed for the rapid, simple, and automated fluorescence analysis of single-cell
suspensions. The VYB also facilitates the absolute quantitation of cell populations
and has a processing rate of up to 10,000 events per second.
Operation of the instrument is extremely simple through the use of the touch screen and
screen menus built into the software. The user has the option of performing simple analyses
pre-programmed into the software using the Express mode or of customizing sample
analysis protocols and automation programs using the Custom mode. Data analysis using a
variety of display options and functions can be performed on the instrument.
The instrument was designed with automation in mind. The VYB employs a robotic needle
arm to acquire cell samples and to apply the sample into the flow cell. The robotic arm
provides the advantage of automation and the ability to sample a specific volume. The VYB
can therefore count an absolute number of cells per mL(error margin ±5%, reproducibility
(CV)<5%). This
also
means
that
multiple
cell populations
can
be
simultaneously
enumerated within a sample after fluorescence staining and analysis.
Automation of cell sampling and analysis can be extended to include the labelling of cells.
With the use of the optional MACS MiniSampler, the automated processing of up to 96
samples is possible facilitating the integration of the instrument into high-throughput
procedures.
2 2. Overview Of The Instrument Parts
Standby light
Storage
solution
Waste
Touch screen
Needle
arm
Washing solution
Running solution
MACS
MiniSampler
Bar-code reader
(behind rack)
Pump syringes
Figure 2.1 Front image of the MACSQuant Analyzer.
Figure 2.2 Symbols and colour coding of fluid container
3. the machine
*Startup
Switch on the VYB by pressing the touchscreen monitor (while it is in standby mode –
you can see a red light next to the screen indicating that it is in standby). The cytometer
will automatically initialize and clean the system, following which the log in screen will be
displayed.
*Shutdown the machine
Click
to select ‘data analysis mode’ or ‘instrument off’ (see next sections for further
information).
This automatically starts a washing protocol that lasts for approximately seven minutes,
which includes an incubation of the washing solution in the fluidics followed by the
flushing of the washing solution and replacement with the storage solution.
3 3. Optics
The VYB is equipped with 3 different lasers, violet (40 mW), blue (50 mW) and yellow (100
mW), and can analyse fluorescence labelled cells using up to ten optical parameters — eight
fluorescence and two scatter channels.
Table 3.1 Summary of compatible fluorochromes and respective channels.
Excitation
Bandpass Filter
Channel
wavelength
(nm)
V1
450/50
VioBlue, CFP, BFP, DAPI, AF405, DyeCycle Violet,
Pacific Blue, BD Horizon V450, Cascade Blue,
eFluor450, Hoechst Dyes
V2
525/50
VioGreen, GFP, PacificOrange, Krome Orange,
Horizon V500, Alexa Fluor 430
488 nm
B1
525/50
GFP, FITC, DyLight488, CFSE, YFP, AF488
Blue
B2
614/50
PI, Lss-mKate, 7-AAD
FSC
561/10
-
SSC
561/10
-
Y1
586/15
PE, Alexa Fluor 555
Y2
615/20
mCherry, tdTomato, Texas Red, dsRed, PE-Texas
Red
Y3
661/20
APC, AF647, PE-Cy5, PE-Cy5.5, mKate
Y4
750 (LP)
405 nm
Violet
561 nm
Yellow
Fluorochromes
PE-Vio-770, APC-Vio770, PE-Cy7, APC-Cy7, APCeFluor780, mPlum, PE-AF750, APC-H7, APC-Alexa
Fluor 750
4 4. Getting Started In The Custom Mode
The Custom mode is designed for advanced flow cytometry users. Administrators and
customer users can use the Custom mode interface to create customized experiments
ranging from sample autolabelling and uptake, through data acquisition, gating and data
analysis, to the generation of print-ready results.
This section is intended to provide the user a quick overview of actions required for a quickstart of the MACSQuant Analyzer in the custom mode.
4.1
Checking the instrument status
The instrument status can be monitored using the status bar and illuminated bottles.
In order to start experiments the VYB should report the status Calibration Ok
The instrument status is reported by the status bar using text and a corresponding colour
code.
Orange: MACSQuant Analyzer in Data analysis mode—the instrument can only analyse data.
The instrument must be placed in acquisition mode to perform a measurement.
Green: MACSQuant in Acquisition mode—calibration was successfully performed one day
ago (time in days since the last calibration is indicated).
Yellow: Cleaning and priming of MACSQuant Analyzer—instrument is not available for
measurement, cleaning in progress.
Grey: Instrument is being initialized – instrument is not available
5 Blue: MACSQuant® Analyzer is processing a sample
The MACSQuant Analyzer is equipped with light-emitting diodes (LEDs), which illuminate
each bottle to indicate the status of the instrument in Acquisition mode:
Green bottle light: The instrument is ready to measure, liquid levels are sufficient, and the
instrument is primed.
Please note that the lasers can take up to 30 minutes to warm-up after performing the
initial instrument priming.
Blue bottle light: The instrument is measuring, liquid levels are sufficient. Blue light is
indicative of normal instrument function during sample processing, or that the instrument is
busy.
Red bottle light: Liquid level error/general instrument error. Red light indicates that the
liquid levels are too low in a particular bottle or that the waste needs to be removed. The
bottle with the blinking red light will indicate which bottle needs to be tended to.
Additionally, a message on the system status in the lower left corner will also report that
that a bottle change is needed. A bottle can be replaced even during a measurement,
although NOT when the instrument is performing a washing routine. It is nevertheless
recommended to replace the waste bottle when the instrument is not processing.
If no LED is illuminating the fluid containers then the instrument is in the Data
Analysis mode and the lasers are not on.
6 4.2
Daily automatic calibration with MACSQuant calibration beads
If you are the first person of the day using the machine you might find a message in the
status bar reporting that the machine must be calibrated.
The calibration is done using MACSQuant beads that consist of two sizes of beads (2 µm
unstained beads and 3 µm beads stained with fluorochromes to emit fluorescence in all 7
channels). You can find the beads in fridge 2, but if you are not familiarized with this
procedure please ask a member of the FACS lab to show you how to do it.
Protocol:
*Prior to beginning calibration, ensure that the single tube holder is correctly attached.
1) The calibration beads can be slightly different from lot to lot which means that the lot
number must associated with the calibration file. This is done by scanning the vial
barcode. Click the barcode icon (
) and present a vial of MACSQuant® Calibration
Beads in front of the barcode reader. To proceed with the calibration process, select
Yes.
2) Follow the dialog box instructions i.e., place an empty tube into the single tube
holder and dispense one drop of beads into it. Ensure that you mix the calibration
beads prior to dispensing.
3) Click OK to commence the calibration process. The uptake needle will dilute the
calibration beads to a total volume of 0.5 mL, following which 150 mL will be taken
up and injected into the flow sheath port for the calibration procedure. During
calibration the gain and trigger for each respective channel will be automatically
adjusted.
When the process is successfully completed, the Status bar should report MACSQuant
ready: Calibration OK. These settings will be automatically saved as the default settings
using the current date.
Calibration results for each channel are presented as dot plots, histograms and as a
tabulated summary on a two-page (two-screen) report. Successful calibration for each
channel is indicated by a green checkmark. To view all calibration dot plots and histograms
7 click Next screen
or Previous screen
. An example of a successful calibration is
shown on Fig. 4.1.
Figure 4.1- Successful calibration of the MACSQuant Analyzer as shown by an array of histograms
(left) and associated summary table (right).
8 5. Quick Guide To The Software Menus
Top menu bar
icons
Tabs
General menus
5.1
Top menu bar icons
Icon
Workspace area
Description
Icon
Description
Folder icon to open Workspaces,
Instrument
Settings,
Experiments, Analysis templates
and/or Data files, depending on
user access rights set by the
administrator.
Activate the Analysis template tool
Click
to
save
Workspaces,
Instrument
settings
and
Experiments, depending on user
access
rights
set
by
the
administrator
Scroll through samples listed in the
samples window
Print.
Backup data.
Delete a region that was created
in a dot plot or histogram.
Activate
scanner.
9 the
reagent
barcode
Draw a region in a dot plot, i.e.,
to define an area of interest.
Open the rack dialog box.
Ellipse,
rectangular
and
polygonal regions can be drawn.
Open the instrument settings dialog
box.
Activate touch screen keyboard.
5.2
Draw a quadrant in a dot plot.
Open help file and manual.
Draw an interval in a histogram.
Switch to Express mode.
Open a new analysis window.
Logout user from session.
Close analysis window.
Main instrument control. Click to
switch between Acquisition mode or
Data analysis mode. The instrument
may also be switched-off using this
button.
Scroll through open analysis
windows in a reverse and
forward direction.
Name of user in the top right
corner, in this example, the
administrator (admin) is logged-in.
General menus
Many of the top menu icons are available in the General menus. In this sub-section you can
find a description of the options exclusive to the General menus.
10 File
Command
Description
Import FCS file…
Import data files in the FCS file
compression format.
Copy…
Copy
Workspaces,
Analysis
templates,
Instrument
settings,
Experiments, Data files and other files
to external media or network drives.
Print…
Print a selected area.
Print all
Print the entire workspace.
Quit
Close MACSQuantify Software – this
option is available to PC users only.
Edit
Command
Description
Undo/ Redo
Undo or redo the most recently
performed action, e.g., undo - create
region.
Copy page
Copy the entire analysis window to
the clipboard. All dot plots and tables
are copied.
User
settings… Create and/or modify user account
settings. Administrator only.
Instrument settings… Modify
the
instrument
settings
comprising Channel, Compensation
and Additional Settings.
Rack…
To edit the sample rack settings.
Reagents…
To open the Reagents dialog box and
modify reagent settings
Options…
To
modify
User,
Experiment,
Instrument and Software options.
Administrator only.
Calibration…
To view and/or modify instrument
calibration settings.
11 View
Command
Description
Hardware
To view the hardware settings
comprising Fludics, Sample uptake
unit, Lasers and detectors, Camera
and System settings.
Experiment table
Provides a tabulated overview of
experiment
details:
Acquisition,
Annotations,
Autolabel
and
Settings.
Mode
Command
Description
Dot plot, Density plot, Click icon to change the presented
Histogram,
Statistic, data format into another format, e.g.,
Text
from a dot plot into a histogram.
Multilayer mode.
View data in a multilayer format.
Switch on Multilayer mode to view all
parent regions on a plot.
Window
Command
Description
New analysis window
Open a new analysis window using
predefined templates.
Clone window
An exact clone/copy of the analysis
window is made. This includes gating
strategies and open data files.
Close/Close all
Close
a
selected
analysis
window/close all analysis windows.
Previous
window/ Sequentially scroll through analysis
Next window
windows, i.e., previous and next
analysis window.
12 5.3
Tabs
Samples tab—displays list of sample files available for analysis with MACSQuantify
Software.
Experiment tab—define experimental parameters for sample acquisition. When using the
software for analysis, users can use the experiment tab to redefine annotations of
previously acquired data files.
Tools tab—find access to some functions such as needle arm calibration. These functions
can only be altered by a member of the FACS lab.
Channels tab—view optical channel settings of a specified data file or instrument settings
file. When using the software for analysis, users can use the channels tab to redefine data
display scales of previously acquired data files.
More information about the different Tabs options can be found in the next section.
13 6. Setup The Experiment:
1) Click the Experiment tab.
2) Select the rack type using the Rack dropdown list.
•
Single samples are processed using the Single Tube Sample Rack
•
Multiple samples (up to 96) are processed by using the MACS MiniSampler in
combination with the Chill 5 or Chill 96 racks (see section 7 for more information
and images of the racks).
•
If you want to keep your samples cold the racks should be pre-cooled for 3–4
hours in one of the FACS lab fridges. Do not chill below 0 OC.
3) Change the filename.
The filename is automatically created by the software using the following nomenclature:
<CRUK><Date (YYYY-MM-DD)>. To change the filename deactivate the File checkbox
and enter a filename into the File field.
4) Create a Folder to save your experiment
Write your LAB name and USER name in the Project box.
5) Select a flow rate: Low, Medium or High.
•
Low: 25 µL/min
•
Medium: 50 µL/min
•
High: 100 µL/min
To select an event-based instead of volume-based flow rate, you can deactivate the
Flow Rate checkbox and select one of the options:
•
500/s
•
1000/s
•
2000/s
6) Select an analysis mode from the Mode dropdown list:
14 •
Sheath consumption during washes between samples: 1700 µL
Washing cycle time: 25 sec
Default option when you open a new workspace. Carry-over below
0.01%
Sheath consumption during washes between samples: 750 µL
Washing cycle time: 12 sec
If you have many samples (and your booking time is short) you can
select this option.
Sheath consumption during washes between samples: 750 µL.
After last sample 2300 µL.
Washing cycle time: 4 sec between samples and 22 sec after last
sample
Be careful when choosing this option. Only part of the needle is
wash between samples and the carry-over may increase, speacially
if your sample is clumpy.
Uptake volume needs to be between 10-50 µL.
•
•
•
Sheath consumption during washes between samples: 5800 µL.
Washing cycle time: 72 sec
If you have clumpy and sticky cells you should select this option.
•
Rare cells can be magnetically labelled and enriched by the MACS
Cell Enrichment Unit. Please see section 11 for further information
Standard
•
•
•
Fast
•
•
•
•
•
Screen
Extended
•
Enrich,
EnrichS,
EnrichS2
7) Enter the Uptake volume (volume of sample that will be collected and run in the
machine)
and
Sample
volume
(total
volume
of
your
sample).
The uptake volume must be between 10 µL and 450 µL and the sample volume must be
a value between 25 and 5000 µL.
Optional: Click the Mix sample checkbox to premix the sample before sample pickup, data
acquisition and analysis.
*The maximum measurement rate of VYB is 30 min for a 96-well plate if you select an
uptake of 10 µL per well and the washing mode screen.
8) Create a new plot window by clicking the new analysis window icon. Nine analysis
templates are available to choose from (Fig. 6.1). Analysis templates consist of a plot
template (new analysis window) and a gating strategy. Analysis templates can be
created post-acquisition or during acquisition in “Live mode”.
15 Fig. 6.1- Nine templates are available to choose.
*Multiple analysis windows can be opened. If multiple analysis windows are open, use
the top menu bar icons to display the previous and next analysis window.
*The size and layout of the dot plot windows are predefined; however, users can
change the properties of each individual plot or histogram using the
icon in the top
left corner. Users can also convert, for example a histogram to a dot plot, counter plot
or text table. For more information see section 7.5.
9) Ensure that reagents, samples and buffers are correctly positioned. Check that the
waste bottle is empty.
*The buffers are stored next to the machine.
*If the waste bottle if full, take it from the rack and add Virkon powder (Virkon is kept
under the sink). The waste should be treated with Virkon for at least 20 min and after
that you can empty the bottle into the sink by LSRIIA.
10) Click Start Measurement, to start acquisition.
*Data is saved from the instant that the sample starts to run. If you adjust
the PMT voltages you MUST click on Clear (in the status bar) to record the
events with the same voltage. Everytime that Clear is pressed the event
counts is reset back to zero.
16 7. Multisample Processing
Multisample processing is accomplished by use of the MACS MiniSampler in combination
with the MACS Reagent Rack 4 and MACS Cooling Tube Racks (sample tube racks). Three
different kinds of sample tube racks are available: Single tube rack, Chill 5 and Chill 96
allowing for processing of up to 96 samples in a single batch (see Table 7.1 for details). The
MiniSampler can be configured to perform measurements from any sample position.
Depending on the user’s instructions, samples can also be automatic labelled with
dyes/antibodies using the reagent rack 4 (more information on section 7.3).
Table 7.1- Overview of the various rack types that may be used with the MACSQuant Analyzer. An
appropriate rack should be used, depending on the sample number and volume.
Rack type
Maximum number of
samples
Single tube
rack
1x5 mL tube
Chill 5
24x5 mL tubes
Rack
Corresponding rack
graphic in the software
Not applicable
Chill 96 rack/
96-well microtiter plate
96 rack
Reagent rack 4
4
Not applicable
17 *The extension of needle arm is calibrated for U-bottom 96-well plate. If you have to use
a V-bottom or a flat-bottom 96-well plate please speak to a member of the FACS lab in
order to re-calibrate the needle arm.
7.1
Setup a sample on a rack
Sample racks are represented graphically by the MACSQuantify Software. All rack positions
are given by coordinates: columns are assigned numbers; rows are assigned letters.
When you open the rack window the different sample positions can be selected according to
the number of samples to be analysed. The selection can be made by clicking the left mouse
button or by touch in screen with your fingertip. You can also select an entire row or column
by right-click in the row letter or column number. When you select a position in the rack a
colour is attributed to that well. Table 7.2 gives an explanation of the different colours
configurations.
Table 7.2- Meaning of the different colours configurations of wells when using a multisample rack.
User action with left mouse
button or fingertip action on
touch-screen
Effect
Details
None
Default open circle indicates no operation: Clear
Single click on circle/single
finger-touch
Closed green circle with orange rim: Sample
selected for measurement. Orange circle indicates
that the sample is selected and any alterations made
to the measurement strategy (e.g. sample name,
labelling strategy, uptake volume, etc.) will only
apply to sample positions with this designation.
Double click on circle/double
finger-touch
Closed green circle: Sample selected for
measurement
None
Closed blue circle: Measurement in progress
None
Closed grey circle: Measurement finished
None
Closed yellow circle: Processing of sample has
commenced, e.g., sample has been labelled and
incubation is underway
18 7.2
Sample Grouping
In certain situations it might be useful to group samples. Sample grouping can be made
before acquisition or afterwards during data analysis.
What is the purpose of grouping samples?
The maximum sample volume that can be acquired in a single step is 450 µL. There are
occasions when the sample size is of course greater; aliquots of the sample must therefore
be spanned over two or more tubes. By grouping these samples, the acquired data will be
consolidated into a single file on the hard drive, which can also be analysed in a single data
file or analysis plot.
How to group samples?
1. Using the Experiment tab, select the desired rack from the drop-down list. Open the
rack window and select the samples for grouping. These sample positions must
be in a column (Fig. 7.1).
2. Click the group button (highlighted by a red box in Fig. 7.1).
3. Click on additional desired rack positions to perform further grouping. Add sample
information as required.
Fig. 7.1- Sample grouping.
19 7.3
Automatic labelling of samples
The MiniSampler allows you to automatic stain the cells while acquiring and you can add up
to 4 different reagents to the samples. You will need one of chill racks in combination with
reagent rack 4.
Protocol:
1. In the Experiment tab select one of the racks and highlight the number of wells
according to the number of samples you have. On the example the A1 and A2 wells
were selected (wells highlighted in green on Fig. 7.2).
Fig. 7.2- Positions A1 and A2 are selected for automatic labelling of samples. The meaning of
the colours assigned for the wells is described on table 7.2.
2. Define the experiment settings for the highlighted wells. To define the position of the
reagent to use during the auto labelling you have to go to the Autolabel tab (inside
the Experiment tab – highlighted in red on Fig 7.3) and click on add. You can also
open the reagent window by selecting Edit>Reagents.
3. Reagents can be entered using the bar code reader or manually using the Reagents
window (Fig. 7.3). You can select reagents from a drop-down list and assign
reagents to a position on the reagent rack. The available options are described
below:
a. Pos: Use this checkbox to assign reagents to rack positions R1, R2, R3, R4, S1, or
S2. R1-R4 positions are located on the rack. S1 and S2 positions are denote as
“Special” positions, where the Running Buffer is taken directly from the buffer
bottle.
Note:The MACS MiniSampler must be correctly installed to view these options.
20 b. Category:
Reagents
are
categorized
according
to
species,
conjugated
fluorochrome and purpose. The current categories follow:
•
Calibration: MACSQuant Calibration beads for calibration of the instrument settings
•
Species and conjugated fluorochrome, e.g., Human – APC, Mouse – PE
•
Isotype control: isotype control antibodies are raised against non-mammalian
epitopes and can be therefore used as a negative control for non-specific binding.
•
MACS Comp Reag. (MACS Compensation Reagents): These reagents are used to
correct the inherent spectral overlap between excitation and emission wavelengths of
fluorochromes.
•
Universal (for generic labelling strategies)
c. Reagents: A drop-down list of available reagents is displayed in accordance with
the selected category.
d. Time: For autolabelling an incubation time is given. The recommended incubation
time is automatically shown in a black font type. You may wish to change the
incubation time using the adjacent arrows; note that non-recommended times will
appear in a red font type.
e. Titer: For autolabelling a recommended label to sample titer is given. The
recommended titer is automatically shown in a black font type. You may wish to
change the titers using the adjacent arrows; note that non-recommended or
changed titers will appear in a red font type.
f.
Order: Signifies the order at which this reagent will be used during cell
processing.
*If your reagent is not in the list you should select Category: Universal. In the
Reagents menu you can select one of the antibodies present in the list or Antibody #1
to #4 or Propidium Iodide Solution.
4. Click Apply to save changes and close the window.
5. The reagents created will be listed in the Autolabel window (highlighted in red on Fig.
7.3). Now you can assign which reagent(s) should be added to each well.
21 Reagent window
Fig 7.3- Automatic labelling of samples. Highlighted in red is the autolabel option. When you click on
add a new window is opened where you can select the antibody you will use, the incubation period
and dilution factor. You can also assign different names to the PMTs by using the annotation window
(highlighted in yellow).
22 8. Working with Regions and Gates
To select a population of cells for further analysis or to obtain statistics on the population, a
“region” can be drawn around it. There are a number of tools for drawing regions and the
icons for these are shown in the table of section 5.2.
8.1
How to create hierarchical gates while acquiring my samples?
‘Gates’ are created within ‘gates’ in order to identify sub-populations of cells.
1)
While acquiring your sample you can click one of the following icons
to
select a geometrical shape for gating. On Fig. 8.1 a polygon region (P1) was drawn to
exclude unwanted debris and select for CD4+ lymphocytes.
Fig. 8.1- A polygon region (P1) was created to select only the cells of interest.
2) To view only the cells within the region P1 in the next do-plot, you must click on the
title of the dot plot. A drop down list will be available where you can select P1.
3) You can continue to draw gates within the population on P2 using the same strategy.
4) In the Sample tab you can visualize your gating strategy and the statistics associated
with the sample, by clicking the “+” signal of the respective sample (see Fig. 8.2).
23 Fig. 8.2- Your gating strategy and the select statistics of the samples can be visualized in the Sample
tab (highlighted in red).
8.2
“NOT” gates
NOT gates are used to eliminate a cell population from analysis. To create a NOT gate you
have to:
1) Draw a region or gate around the population to be excluded from analysis.
2) In the Samples menu, right-click on the region you just selected and select Region
properties from the drop-down list.
3) Check the box Not. This region is now negated from analysis. Click Apply and OK.
8.3
Stopping gate and live gate
All data are acquired and saved by the so-called Stop gate. However, a Stop gate used in
combination with the ‘Events’ option instructs the software to acquire data from the entire
analysis window until a pre-defined number of events are acquired within the Stop gate (i.e.
a gate or region that is defined as the stop gate).
How to create stopping and live gates?
In the bottom part of the experiment tab you can select a second tab called Settings. Here
you can define a stopping or a live gate (Fig. 8.3).
24 Fig. 8.3- Region P1 was defined as a stop Gate. When 10,000 events are acquired within region P1,
data acquisition will automatically stop and all acquired data (i.e. the entire analysis window) will be
saved.
* Be careful if you choose the live gate option, because only events within a live gate
are acquired and saved. All data outside of the gate will not be saved! Live gates are
useful when large datasets are being acquired. Since only data in a single gate are
saved the subsequent data file is smaller. As a norm, however, we recommend to
acquire and save all data.
25 9. Changing the Properties of a Plot, Histogram or Text Table
The properties of a dot plot, density plot, histogram, and statistic or text box can be
changed by clicking in the icon
9.1
Tab
option
View
.
Overview of the “Properties” settings for dot plots, density plots and
histograms
Category Property
Data
All
Description
All events are displayed on a dot plot, density plot or
histogram.
50%, 25%, 10%, 5%, 2% or 1% percentile values of the
total events can be displayed on a dot plot or density
Percentile
plot. This is useful when too many events have been
acquired and the displayed plot/histogram is saturated.
Fixed
number
Axes
X axis
Y axis
Options
Region
functions
All
A fixed number of events can be displayed on a dot plot,
density plot or histogram. Numbers can be entered
directly into the field or by using the arrows.
The y- and x-axes scales of a dot plot and density plot,
or the x-axis scale of a histogram can be configured as
follows:
As required – automatically configured
lin – linear scale
log2-5 – logarithmic scales
hlog – hyperlog scaling
All regions (“gates”) are shown on the chart.
This
Only a select region (“gate”) is shown on the chart.
None
No regions (“gates”) are shown on the chart.
Regions
Regions (“gates”) can be displayed (
by selecting or deselecting a region.
) or hidden (
Functions for a region can be displayed (
) or hidden (
) by selecting or deselecting the function.
The following functions can be changed for plots and
Functions histograms:
Name: Region name e.g. “P1”
%-#: Events in selected region are displayed as a
percentage of the total number events in the current
gating strategy.
26 )
%-T: Events in selected region are displayed as a
percentage of the total number of acquired events.
Count: The actual number of events occurring in the
selected region is displayed. This can be displayed either
as count/mL or count /µL.
9.2
Overview of the “Properties” settings for the Statistic option.
Tab
option
Category
Property
View
Options
Header
Check the Header box to include this information in the
text box.
Table
Check the Table box to include this information in the
text box.
Column headers (titles) of the table can be displayed in
two formats: “shown using annotations” and “shown
using channel names”. Annotations are defined by the
user. To exempllify, “FL1-A” could be annotated as
“VioBlue-A”.
Region
functions
Regions
Description
The settings for regions and functions are identical to
those for dot plots.
Functions
Feature
function
Features
The recorded time, scatter, and fluorescence data
acquired from each channel may be displayed using the
Features setting. The data shown will be dependent on
the selected regions (see Region functions). Features
can be displayed ( ) or hidden ( ) by selecting or
deselecting a feature using the left mouse button or by
touching the display with your fingertip.
Statistics (or “Functions”) for each selected Feature
Functions
can be displayed ( ) or hidden ( ) by selecting or
deselecting a Function using the left mouse button or by
touching the display with your fingertip. The following
statistical functions may be displayed: Mean; StdDev
(standard deviation); CV (coefficient of variation); Min
(minimum); Max (maximum); Median and Modal values.
27 10.
Compensation
The aim of compensation is to account for spectral overlap of fluorochromes into adjacent
detectors. With all flow cytometers, problems can be encountered when using multiple
fluorochromes because the emission of each fluorochrome can “spill over” into the detectors
of others. For further explanation on the principles of Compensation, please speak to any
member of the FACS Lab.
For proper compensation, the use of single-stained controls is absolutely necessary. These
single stains can be cells, stained compensation beads or antibody-capture compensation
beads, but there are several points to take into consideration when setting up and choosing
single-stained controls for compensation. Here, you can find some general guidelines for
proper compensation:
•
Compensation should be performed using the same fluorochrome that will be
detected in the experimental panel. This means that if FITC is used in the
experimental staining panel, FITC must be used to set the compensation.
Do not
substitute with another fluorochrome that is detected in the same channel, such as
GFP or Alex-fluor488.
•
If utilizing a fluorochrome conjugated to a different antibody for setting the
compensation adjustments, the fluorescent intensity level of this staining should be
as bright as or brighter than the signal from the experimental panel.
•
All changes to voltages of fluorescence PMT detectors must be made prior to adding
compensation adjustments. Changing PMT voltages after compensation will change
the spill over detected and will require reevaluation of compensation values.
•
Compensation can be set using antibody-capture compensation beads.
However,
remember to not change fluorescent PMT voltages after setting the compensation.
•
If using tandem fluorochromes, the exact same lot from the same manufacturer
must be used to set compensation adjustments. In these instances, compensation
beads or control cell lines can be useful.
28 •
Proper compensation should not be performed using visual inspection of fluorescence
intensity. Statistical analysis of the median fluorescence intensity for the ‘spill over’
channel must be used to set the adjustments.
•
Using hlog scaling for data display is preferred when visualizing compensated data.
The option is available to select in the drop-down list next to each detector channel.
It is recommended that the software automatically calculates the appropriate compensation
settings by using the automated compensation matrix and the compensation program
(matrix method). However, more advanced flow users may wish to adjust these settings
manually using the sliders. The MACSQuantify Software allow the user to set compensation
in the following ways:
1. Automated compensation using the Express program CompensationMultiColors.
2. Manual compensation while acquiring using the fluorescence median technique.
3. Recompensation offline within MACSQuantify Software after data acquisition.
10.1 Automated
compensation
CompensationMultiColors
using
the
Express
program
Choosing this option, the instrument will perform fully automated compensation on all
channels at one time. You will need to have single-stained controls representing all
fluorochromes to be used in the experimental staining panel, prepared in FACS tubes or in a
96-well plate. Ensure that there is a comparable positive and negative population for setting
compensation. Your negative control can be present in your tube with the positive
population (a mix of both) or you can have a separate tube only with the negative
population.
Protocol:
1. Run all your single-stained controls and adjust the PMT voltages. The optimum
voltage set up is one where the signal from positively stained populations can be
best separated from negative/unstained populations, with negative populations being
positioned above the electronic noise and at the same time allowing dim populations
to be distinguishable above background. The voltages should be balanced against the
other fluorochromes used so that spill-over can be compensated out.
29 2. Select one of the two available racks from the Experiment tab (Chill 5 rack used as
an example on Fig. 10.1). In the large Chill 5 window, choose the appropriate
number of positions to match the number of samples for compensation.
3. Click on the Group button at the bottom of the window. Each of the selected rack
positions should now be labelled with the number ‘1’.
4. Choose Express from the Settings tab in the Experiment section and select Setup in
the upper drop-down list and CompensationMultiColors from the lower list.
5. To set the fluorochrome for each rack position highlight the respective circle in the
rack window. Only one circle must be highlighted at a time. In the Sample ID field a
drop-down list will be available where the representative fluorochrome can be
chosen.
Fig. 10.1- In this example nine samples were selected and grouped. By clicking on each well at the
time you can assign the respective fluorochrome on Sample ID drop-down list.
*Each fluorochrome name in the drop-down list represents the respective detection
channel. For example, FITC represents the channel B1. When using another B1–
compatible fluorochrome, like Alexa-fluor 488, FITC must be selected from the list.
*The position blank is a sample with unstained cells/beads used as a reference for
negative cells. The sample PI is necessary when propidium iodide (PI) will be used for
dead cell exclusion. It is used for compensating all fluorescence channels against the
channel B2. This ensures that there is no fluorescence spillover into the PI staining. The
sample should be unstained cells. The addition of PI is not necessary.
30 6. Place samples in proper rack positions and click the start measurement icon.
7. After prompting by the software, draw a region around the population of interest in
the scatter plot and click on the Continue button.
8. At the end of the process enter the desired name for the instrument setting file in
the Save window and click the Save button.
10.2 Manual compensation
median technique
while
acquiring
using
the
fluorescence
In this option you can manually compensate your samples while acquiring using the
fluorescence median technique. This is done by drawing gates around the positive and
negative populations and comparing the median fluorescence of the two. For example, if
you are compensating FITC out of PE, the median fluorescence of the double negative
population will be X. The FITC+PE- cells have no PE and should therefore give the same
median PE signal as the double negative (X). However, because of the spectral overlap of
the FITC signal into the PE channel, the FITC+PE- population has an increased median PE
fluorescence of Y. To correctly compensate this sample you simply increase the amount of
compensation applied until Y=X. You just have to imagine a line in the centre of the positive
population that needs to be lined up with the centre of the negative population.
Protocol:
1. Select one of the nine available display templates that contain a table.
2. Run all your single-stained controls and adjust the PMT voltages.
3. Open the 8x8 compensation matrix by clicking the instrument settings icon and
selecting the compensation tab.
4. Place the first single-stained tube (e.g FITC-stained cells) into the single tube holder
and start acquisition.
5. When events start to appear on the plots pause the measurement by right-clicking
the stop button and choosing pause.
31 6. Draw a scatter gate around the population of interest within the FSCvSSC dot plot.
This will be P1. Display only events in P1 in the dot plot that you will use to
compensate your sample, by selecting P1 from the drop-down menu of the plot
header. Draw regions around the positive (P2) and negative (P3) populations.
7.
In the statistics table, format properties under the “i” button. Select to display P2
and P3 regions under the region functions. Select to display the median of all the
spill over fluorescence channels (e.g. For FITC spill over, display V1, V2, B2, etc.,
although not all require compensation).
8. Resume measurement by clicking on the pause button. Enter values in the box
within the compensation matrix by using the associated slider. For example,
compensating PE signal from the FITC detection channel, the seventh box down
within the third column (PE column) should be adjusted. Values should continue to
be adjusted until the median fluorescence values for the positive and negative
populations are equal for the spill over channel.
9. Adjust values for other spill over channels as necessary. Once compensation is
adjusted for this fluorochrome, repeat for all additional fluorochromes.
10.3 Recompensation offline within MACSQuantify Software after data
acquisition.
Compensation or adjust the compensation automatically calculated can be performed on
any file acquired.
Protocol:
1. Choose the file from the sample list.
2. Right-click on the file and select Apply instrument settings (see Fig. 10.2).
32 Fig. 10.2- To apply the settings (PMT voltages, threshold, etc) associated with a recorded file right
click on the sample and select “Apply instrument settings”.
3. Open a display template by selecting the new analysis window icon and add the file
to the template.
4. Set up to visualize the first fluorochrome requiring compensation (e.g. FITC) in the
histogram/dot plot.
5. Open the compensation matrix, by clicking the instrument settings icon and selecting
the compensation tab.
6. Draw a scatter gate around the population of interest within the FSCvSSC dot plot.
This will be P1. Display only events in P1 in the histogram plot, by selecting P1 from
the drop-down menu of the plot header. Draw regions around the positive (P2) and
negative (P3) populations.
7. In the statistics table, format properties under the i button. Select to display P2 and
P3 regions under the region functions. Select to display the median of all the spill
over fluorescence channels (e.g. for FITC spill over, display V1, V2, B2, etc.,
although not all require compensation).
8. Enter values in the box within the compensation matrix by using the associated
slider. Once the value has been entered into the matrix, select the file to be
recompensated from the sample list.
33 9. Right click on the file and choose, recompensate. A new file will be created with an
underscore (_) before the file name.
10. Values should continue to be adjusted until the median fluorescence values for the
positive and negative populations are equal for the spill over channel.
11. To avoid creation of multiple files, continue to apply the recompensation to the
original file. This will rewrite the second file (with the ‘_’ before the file name) and
changes will be immediately visible on the analysis page.
34 11.
Pre-Enrichment
Instrument
of
the
sample
with
MACSQuant
The inclusion of the MACS Enrichment Unit within the system permits the magnetic
enrichment of cells in situ prior to fluorescence analysis. The Enrichment Unit allows the
possibility to reduce the number of cells analysed to characterize the rare cell population of
interest. This is particularly useful for the analysis of cells present in low abundance, such as
stem cells, dendritic cell subsets, or natural killer cell subsets.
11.1 Use of MACS Technology for pre-enrichment with the MACSQuant
Column
In a first step, surface antigens are magnetically labelled in a highly specific manner with
monoclonal antibodies coupled to MACS MicroBeads. This is followed by fluorescent labelling
with necessary immunophenotyping antibodies. After magnetic and fluorescent labelling,
the cells are loaded onto the MACS Column placed in the magnetic field of a MACS
Enrichment Unit. Non-labelled cells are gently washed away from the labelled cells that are
retained within the column’s magnetic field. The enriched fraction is then eluted from the
column when the magnetic field is disengaged and flow directly to the flow cell for
measurement.
* Cells have to be labelled magnetically and fluorescently since they will be
immediately measured after enrichment and cannot be labelled after that.
11.2 Properties of the Column
Capacity—retains up to 5x106 magnetically-labelled target cells
Life-span—3 months
Sample volume in Experiment Tab = Volume of sample to enrich on MACSQuant
Column—maximum of 5 mL
Uptake volume in Experiment Tab = Volume of eluted fraction to measure on
MACSQuant Instrument—maximum 450 µL.
35 11.3 Enrichment Modes
Enrich
Process
description
EnrichS
EnrichS2
Designated sample volume is loaded onto MACSQuant Column.
Negative fraction is washed away to waste.
Positive fraction is eluted and directed to flow cell.
Min/max sample 25 mL/5 mL
volume
25 µL/5 mL
25 µL/5 mL
Cell separation
rate
0.5 mL/min
0.25 mL/min
Cell washing rate 1 mL/min
1 mL/min
0.5 mL/min
Volume of buffer 4.66 mL
4.66 mL
2.66 mL
Elution rate
37.5mL/min
25 mL/min
37.5 mL/min
Elution volume
450 mL
450 mL
450 mL
0.75 mL/min
36 12.
Opening and Saving Data Files, Experiments and
Instrument Settings
This section describes how data files can be opened and saved in Custom mode. Data
files may be stored to and therefore opened from a Public, Private, or External file
location:
•
Public files are located on the local hard drive of the instrument and are accessible by
all users.
•
Private files are located on the local hard drive of the instrument and are only
accessible by the logged-in user account. Currently all files are saved as private files
in the administrator account. No password is required.
•
External files are located on an independent file storage device, which is connected
to the instrument via the USB port i.e. a memory stick.
The window for saving
and opening
data files is composed of the following tabs:
Tab
Description
The Workspaces tab allows users to save an entire workspace, which is
composed of instrument settings, experiment and reagent definitions, and an
analysis template with accompanying data.
Instrument settings are compensation and calibration parameters for the
instrument.
Experiment definitions can be saved for future use. Reagent type and
corresponding Reagent Rack 4 positions, sample rack type and corresponding
Chill Rack sample positions, the analysis mode and sample processing
definitions (e.g. labelling strategy) comprise experiment definitions.
Analysis templates are predefined analysis layouts for data acquired by the
MACSQuant Instrument. The templates are creating by defining a gating
strategy with associated plots, histograms, tables and statistics. Users can
customize and save templates for reuse.
Data files can be saved to a Public, Private, or External file location by all
users. MACSQuant Data Data (.mqd) is the standard file handling format,
however, the software can also create Flow Cytometry Standard (.fcs) file
types.
37 13.
Export Files, Templates,
Sample Information
Instrument
Settings
and
13.1 Transferring .fcs files, templates and instrument settings using the
copy function
Currently you can export your files from VYB to the PC placed next to it. This PC is
networked and you can access your lab server. To export your files you have to:
1. Go to File > Copy. A window will open where you have to write your LONRES
credentials. In the user name you have to type lonres\ and your username (eg.
lonres\cervei01).
After the logging in, a new window will open (Fig. 12.1) were you can select the files
to be copied. The .fcs files are in the data files sub-menu. Note that the PC will be
selected in the drop-down list (highlighted in red on Fig 12.1).
Fig. 12.1- Copy window. Here you can select the relevant files to be copied to your USB
device. The .fcs files are on the data files sub-menu.
2. Inside the private folder you can find the folder where your .fcs files are saved.
Select the relevant files and click on copy.
38 13.2 Export Sample Information As An Excel Spreadsheet
It is possible to export the sample list to an excel table. The sample list table is a summary
of all samples with corresponding statistics (number cells/ml or µl, percentage of cells within
a gate, etc.).
To export a sample list:
1. Click the Samples menu.
2. Highlight the sample files that must be exported. Right-click and select Export
sample list.
3. Configure the Export sample list box as follows:
Options tab:
•
Clipboard: check box if you want to export data to the windows clipboard.
•
File: check box if you want to export data to a Microsoft Excel file (xls).
•
Location: files can be saved to a Public or Private location.
•
Name: enter the filename here, taking care not to delete the Excel file extension
“.xls”.
•
Conversations – the respective boxes to:
o
Convert comma to point: excel does not support decimal points being
assigned a comma “,”.
o
Transpose rows and columns: the columns and rows of the export table (e.g.
Excel sheet) are inverted.
o
Reverse samples: the order of the samples (e.g. 1,2,3…etc) are reversed.
39 Region tab:
•
Select the gates/regions for export using the Regions box. Similarly, use the
Functions box to select/deselect region functions for export.
Feature tab:
•
In a similar manner, select/deselect Features (i.e. Channels) and Functions (i.e.
statistics) for export.
4. After configuring the export options, click OK. The file is exported to an Excel file
and/or the clipboard.
5. The file will be available to copy under File > Copy > Other Files (see Fig. 12.1).
40 14.
Symptom
Troubleshooting
Cause
Touchscreen Instrument
remains
may not be
black
powered up
Air bubbles in
the system
Solution
1) Check if the power cord is plugged in correctly and the
power supply is switched on.
2) If the device is powered-up and the touchscreen remains
dark, switch-off the device, wait 5 seconds, and switch on
again.
Perform a rinse program (click the teardrop symbol) or a
clean program (right click in the teardrop symbol)
Check for leakage within the fluidics and tubing system.
Check that the tubes connected to the bottle closures are
tightly sealed.
Leak in the
fluidics system Ensure that the pre-filter is devoid of air (ask a member of
the FACS lab to show you how to do it).
No events
displayed
Excessive of
debris is
present in
the
acquisition
Perform a rinse program
Needle is not
well calibrated
Ask a member of the FACS lab to test the needle calibration
and calibrate it if necessary.
Needle is
blocked
Perform a Flush program (right click in the teardrop
symbol). The flush program will perform a backflush to
remove or dislodge any blockages in the sample uptake
system with the washing solution. The process takes 16
min.
Air bubbles in
the system or
leak in the
fluidics system Check for a solution in the previous rows
or needle
blockage
41