Partec CyFlow space User manual

CyFlow®space
Instrument Operating Manual

Introduction to CyFlow
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 2/26
Table ofcontents
Introduction to CyFlow®space.......................................................................................................................3
Typical Steps of Particle Analysis..................................................................................................................4
Operating Basics..............................................................................................................................................5
Switching on the CyFlow®space.................................................................................................................5
Multi laser Measurements ..............................................................................................................................6
Starting a Measurement.................................................................................................................................7
Switching off the CyFlow®space.................................................................................................................8
True Volumetric Absolute Counting –Overview ............................................................................................9
Performing True Volumetric Absolute Counting...........................................................................................10
Optical Standard Setup –The Parameters ..................................................................................................11
Optical Flow Geometry.................................................................................................................................12
Instrument Settings.......................................................................................................................................12
The Parameter Setup Dialog Box: Pulse Height, Area and Width...............................................................13
Trigger ..........................................................................................................................................................14
PMT High Voltage, Gain, and Log Amplification..........................................................................................15
Sample Speed..............................................................................................................................................16
Threshold: Lower Level (L-L) .......................................................................................................................17
Appendix.........................................................................................................................................................18
Installation Requirements.............................................................................................................................18
Instrument Setup –Overview.......................................................................................................................19
Instrument Setup –Step by Step .................................................................................................................20
The Flow Cuvette .........................................................................................................................................21
Maintenance and Service.............................................................................................................................22
Transport and Storage..................................................................................................................................22
Disposal........................................................................................................................................................22
Laser Safety .................................................................................................................................................23
CyFlow®space - Technical Specifications ...................................................................................................24
For in vitro diagnostic use with Partec recommended IVD reagents.
The Partec CyFlow®space flow cytometer complies with the European IVD Directive 98/79/EC and is
therefore marked with the CE sign.
© 2010 Partec GmbH
Otto-Hahn-Str. 32
D-48161 Münster
Germany
IVD

Introduction to CyFlow
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 3/26
Introduction to CyFlow®space
What...
… is the Partec CyFlow®space?
… are the applications for which the
CyFlow® space can be used?
… topics are covered by this manual?
… other manuals are available?
... should I know before operating the
CyFlow®space?
The Partec CyFlow®space is a fully equipped
desktop Flow Cytometer (FCM). It features a
modular optical concept which allows using up to
three different laser light sources and between
one and nine optical channels (parameters). The
CyFlow®space allows easy adaption of the optics
to any application by simple exchange of optical
filters and mirrors.
The CyFlow®space runs with a standard PC or
notebook. Data acquisition, instrument control,
and data analysis are controlled and performed by
the FloMax®software.
Together with the software, the CyFlow®space
offers automation for routine use and flexibility for
research use for practically any flow cytometric
application. The applications cover e.g.:
Routine and research Immunophenotyping,
Blood Cell Analysis, HIV monitoring
Leukocyte Counting / Rare Event Analysis
Microorganism Analysis
Fermentation Control
Particle Concentration Analysis with the
feature «True Volumetric Absolute Counting»
Particle Size and Fluorescence Distribution
Analysis
The CyFlow®space Instrument Operating Manual
covers the basic operation and maintenance of
the CyFlow®space instrument. This manual does
not cover details related to the software. Different
software operating manuals are available.
FloMax®–Acquisition and Instrument Control
covers instrument control and multiparametric
data acquisition.
FloMax®–Data Analysis covers all aspects of
on- and offline data analysis.
Application Notes and Service Manuals are
available to get started. They contain hints to
achieve the best results.
This manual assumes you have basic knowledge
about flow cytometry. In the best case a well
experienced "flower" is around - so let her/him
help you. Basic books are available about flow
cytometry which may help you as well (e.g.
Howard M. Shapiro, Practical Flow Cytometry.
Wiley 2002)

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 4/26
Typical Steps of Particle
Analysis
1. Cells are separated in a suspension and
stained with fluorescent markers.
2. Light from the laser(s) excites cell –bound
fluorophores and is scattered by the cells.
3. Fluorescence signals are displayed and
analysed in histogram- and/or dotplot-
diagrams.
1. Preparation and Staining
For flow cytometric analysis, the cells (or other
particles) must be separated from each other in
an aqueous suspension. Typically, if not
generating sufficient optical signals by
themselves, the cells are then labelled by staining
with one or more fluorescent dyes. The
fluorescent molecules bind to the cell substance
of interest, e.g. CD4- and CD45-antibodies bind
cell surface antigens of leukocytes. The quantity
of labelling cells with a specific dye is proportional
to the number of antigen molecules of a single
cell. A good preparation with suited reagents is
the requirement for a precise analysis. A check of
staining in a fluorescence microscope can un-
reveal preparation problems.
2. Flow Cytometry Analysis
While passing through a flow cuvette one-by-one,
the cells are individually illuminated by the light
spot of the laser. Due to the excitation, the dye
molecules emit fluorescence of characteristic
colour (emission wavelength spectrum). This
fluorescence light is separated into colour ranges
by means of optical filters. The intensity of each
colour range is analysed for each single cell.
Besides fluorescence, the intensity of light
scattered by each cell can be measured. Scatter
light is measured in forward direction from the
light source (forward scatter, FSC) and sideward
direction (side scatter, SSC). The scatter intensity
is a measure of cell size and morphology. Scatter
light can be used to identify a cell before
analysing its fluorescence, but a cell can also be
identified by fluorescence before analysing its
scatter properties.
3. Realtime Data Processing and Results
The light intensity of each parameter, and with it
the quantity of substance of interest, is assigned
to one of up to 216 or 65536 quantity classes
(channels). The classification is performed in
realtime, while the cells are passing the flow
cuvette. Single parameter histograms show the
number of cells within the channels. Two
parameter dotplots show the correlation between
two cell properties.
4. Absolute Cell Counting
Since the CyFlow®space analyses all cells
passing through the flow cell while precisely
monitoring the fluid volume of the sample, it
allows volumetric counting during the analysis, i.e.
the determination of concentration of any cell
subpopulation. Cell subpopulations can even be
defined and their concentration analysed at a later
date, after reloading data from a file.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 5/26
Operating Basics
Switching on the CyFlow®space
Notes
As sheath fluid you may
use optimized Partec
Sheath Fluid or prepare it
yourself.
To prepare sheath fluid,
fill destilled water into a
glass bottle or an
Erlenmeyer flask (refer
to Application Note:
”Sheath fluid pre-
paration”), through a
small size filter mesh
(< 0.2 µm). Degas the
water by connecting it to
vacuum (e.g. a water jet
pump). Place the bottle
onto a heated stirrer for
about 15 minutes:
Remember: The smaller
the particles in your
analysis are, the more
critical clean sheath fluid
becomes!
It is recommended to
replace the sheath fluid
at least once a week or
before any daily use.
For information how to
work with Windows®
please consult the
manual "Introducing
Microsoft Windows" or
books available in your
local book shop.
1. Check SHEATH and WASTE bottle
Make sure SHEATH bottle is filled with no more than
1600 ml of Sheath Fluid and is closed tightly with the
screw top. Tilt sheath bottle in order to release air
bubbles trapped in the yellow in-line filter!
Make sure the WASTE bottle is empty.
2. Switch on the instrument
The CyFlow®space Flow Cytometer is operated with 100
- 240 V AC.
Switch on main power at the left side of the instrument.
The 488nm laser has its own power button closeby,
please also switch on the 488 nm laser.
All other lasers are software controlled (for details please
refer to next page)
3. Control of the 488nm laser output power (for 200
mW high power laser only)
The 200 mW high power laser can be adjusted in its
operation power between 50 and 200 mW. For power
selection please doubleclick the icon shown on the left
(488-200)
To change selected laser power please type in:
p=value enter
To inquire about actual selected laser power type in:
?p enter
3. Switch on peripheral devices
Switch on printer.
4. Switch on computer
Finally, switch on computer by Computer Power switch.
After some ten seconds, the display shows the
Windows®desktop.
5. Start instrument operating software
Move mouse pointer (arrow on display) onto FloMax®or
other instrument control program icon.
Doubleclick on the icon with left mouse button.
- after a few seconds, the display shows the FloMax®
Welcome window. By confirming with “OK” the CyFlow®
space is initialized and the last used instrument settings
are loaded.
The display shows empty histograms.
6. Cleaning
Use speed 4. Run 1.6 ml Partec Cleaning Solution until
sample tube is empty. Activate CLEAN. Run a second
tube with Partec Sheath Fluid for 2 minutes.
Now, the CyFlow®space is ready for acquisition.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 6/26
DANGER
Warning: Biohazards!
The waste may contain biohazardeous and cancerogenic material from the samples (infectious cells, dyes).
To minimize biohazards, fill about 25 ml of Partec Hypochlorite Solution into the waste container. When
emptying the container, be sure not to come in contact with the fluid. In case of accidental contact, wash your
skin throughoutly with soap and disinfectant.
Multi laser Measurements
Notes
Alignment of the lasers
should only be done by
Partec representatives
or by your local
distributor.
The CyFlow®space Flow Cytometer can be equipped
with up to 3 laser light sources according to individual
customer demands. Actual setup of your instrument may
therefore vary. For questions concerning the laser setup
of this instrument please contact Partec or your local
distributor.
In order to minimize crosstalk between multiple light
sources, laser beams from different lasers are focussed
on different positions (spots) of the flow cuvette. The
CyFlow®space Flow Cytometer supports either 2 laser
spots (when employing FloMax version 2.6 or 2.7) or 3
laser spots (when employing FloMax version 3.0). The
blue 488 nm laser which is the leading laser of the
CyFlow®space is always located at spot 1. Additional
lasers if present are located on spot 2 and spot 3.
While the blue 488 nm laser is switched on with the
button on the left side of the instrument all other lasers
are controlled by software:
Select “Acquisition” from the upper command line and
click on Laser No. 2 and / or Laser No. 3 as required. The
tick indicates that a laser is switched on. Laser no. 2
would be a red laser (if present), laser no. 3 would be a
UV or violet laser (if present).
Laser status will automatically be saved upon creation of
Instrument settings files (please refer to the manual
FloMax®–Acquisition and Instrument Control).
In the standard configuration all particles pass through
laser spot no. 1 of the 488nm laser –the leading laser –
first and then through the laser(s) at spot no. 2 and no. 3
(if present).
Therefore, all parameters derived from a laser located at
a spot other than no. 1 require a time delay. Please refer
to Parameter Setup Dialog Box for details (Fig. 5, page
13).

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 7/26
Starting a Measurement
Notes
See FloMax®- Acqui-
sition and Instrument
control manual on how
to change instrument
settings.
When removing a tube
from the sample port,
the BioSafety system is
activated to prevent
dropping and
contamination by
hazardeous material.
Simultaneously, a
cleaning cycle is
performed to avoid
cross-contamination
between individual
samples.
or
or
1. Make sure your flow cytometer is ready for
analysis and the operating software is prepared for
the measurement.
2. Prepare sample according to the Application
Notes and preparation procedures, resp. Use not
less than 830 µl for true volumetric absolute
counting and not more than 2.8 ml. For smaller
amounts of sample use Partec “Small Volume
Sample Tubes” (Product no. 04-2010, however
these tubes are not compatible with the True
Volumetric Absolute Counting option, page 10).
3. Insert sample tube onto the sample port until
you recognize a "click". The sample should be fully
mounted within a second.
Now the measurement (acquisition) starts
automatically –the operating software indicates the
Prerun, Stabilize, Run and Count status.
Instrument status is also highlighted by LEDs on the
front side of the CyFlow®space.
In the Prerun phase, cells are quickly transported
to the position of analysis, the flow cuvette.
During the Stabilize phase, the system waits a
given time for the flow to reach slow speed required
for analysis.
In the Run phase, cells are analysed and classified
into histograms on the display in realtime.
In the Count phase, cells are counted for a given
volume. Do not change instrument settings in the
count phase.
After the count phase, the acquisition finishes
automatically. To finish the acquisition before this
manually, click the end icon or end button, or simply
remove the sample tube from the sample port.
The BioSafety cycle is started.
Status changes to Clean and then Ready.
To restart an acquisition without removing the
sample tube, click onto start.
4. To save results, click on the disk icon.

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 8/26
Switching off the CyFlow®space
Note
Cleaning
Final cleaning with
sheath fluid is a must to
avoid clogging of the
flow cuvette.
or
Make sure data from the last acquisition have been
saved.
1. Clean the flow cuvette and connecting tubes
Use speed 15.
a) Daily claning procedure:
Run 1.6 ml of Partec Cleaning Solution (Product no.
04-4009) until sample tube is empty. Run 1.6 ml of
Partec Decontamination Solution (Product no. 04-
4010) for one minute. Press CLEAN button once to
start manually a cleaning cycle. Run 1.6 ml Partec
Sheath Fluid (Product no. 04-4007) for two minutes.
Press STOP.
b) Weekly cleaning procedure:
Run 1.6 ml of Partec Decontamination Solution.
Pinch the sheath fluid tube for 5 seconds. Stop the
instrument while keeping the tube pinched. Incubate
for 15 minutes. Re-start the system by pressing
START and let it run to the end. Run the system with
1.6 ml Partec Cleaning Solution until the sample
tube is empty. Run 1.6 ml Partec Sheath Fluid for
two minutes. Press STOP.
2. Protect the sample port
Leave the final sample tube connected to the
sample port –this avoids drying and cristallizing of
any remaining material.
3. Quit the operating software
Click onto the close button [X] in the upper right
corner of the window (or select File...Exit in the
menu).
4. Shut down the computer
Click with the left mouse button onto the Windows®
"Start" button in the taskbar - the Windows menu is
displayed.
Click "Turn Off Computer..." - the shut down window
appears.
Click "Turn Off" in the Shut Down Window.
5. Switch off printer
6. Switch off instrument
Switch off main power and blue laser at the left side
of the instrument.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 9/26
True Volumetric Absolute Counting –Overview
Analysis of concentrations of samples, loosely
called Absolute Counting, can be of significant
interest for medical diagnostics or monitioring the
status of cells in cell cultures or biotechnological
processes.
Traditional Flow Cytometric Techniques for
Absolute Counting
i) Dual Platform Technique
To determine the concentration of a sample flow
cytometers (FCM) are frequently used in
combination with Hematology Counters.
Disadvantages:
Two instruments required
This technique relies on the assumption that
the number of cells seen by the FCM and
counted by the Hematology Counter are
identical.
ii) Single Platform Technique with Reference
Beads
An alternative method employes reference beads
of known concentration which are added to the
sample of unknown concentration.
Disadvantages:
Depends on the accuracy of the specified
beads concentration
Constant running costs for the reference
beads
Partec True Volumetric Absolute Counting
To overcome the drawbacks of the Dual- and
Single-Platform Technique with reference beads,
Partec intruments additionally offer an alternative
way of absolute cell counting, which is based
directly on the basic definition of a concentration
by precisely counting the number Nof cells
suspended in a purely mechanically defined
volume V.
i) Precise Counting: Determination of N
For True Volumetric Counting the precise
detection of cells is an essential. This requires fast
recognition and analysis of the events by
electronics and computer. All Partec instruments
are specifically designed to minimize counting
losses by providing direct connection between
computer and electronics, which avoids dead-
times involved in traditional FCM designs and
instrument interfaces. This reduces the probability
of a counting loss for typical event rates below
2%.
ii) Electrode-Principle: Determination of V
The method for True Volumetric Absolute
Counting supported by the CyFlow®space is
based on the precise measurement of a fixed
sample volume by means of two electrodes.
Fig. 1: True Volumetric Absolute Counting by the
Electrode Principle.
During analysis by the CyFlow®space, the
sample liquid loses contact first from the upper
and then from the lower electrode. These events
trigger a START and a STOP signal, respectively
which is stored by software.
The volume Vof sample liquid analysed between
the START and STOP signal is physically defined
by the distance of the two electrodes and the
diameter of the sample tube. The analysed
volume is exactly 200 µl (= counting volume).
Effects of the sample meniscus in the tube are
eliminated because the START and STOP
electrodes are arranged symmetrically. The
sample conductivity does not influence the
volumetric measurement, as long as the fluids can
be detected electrically.
In some cases, larger or smaller counting volumes
Vare advantageous, either to improve the
counting statistics (e.g. for rare events < 10/ml) or
to decrease the analysis time. This can be
accomplished by replacing the standard 200 µl
volumetric sample port by one with a different
volume (Please contact Partec or your local
distributor for details).
c= N/ V,

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 10/26
Precision and Reproducibility
Precision and reproducibility of the Partec
instruments can be demonstrated and checked
with Partec CountCheck beads (Product no. 05-
4010).
Benefits of True Volumetric Absolute Counting
Partec instruments can perform traditional Dual-
and Single-Platform counting and unique True
Volumetric Absolute Counting, the latter offering
unique benefits:
i) Precision
A high precision of better than 5% is guaranteed
by precise counting and mechanical volume
measurement. The counting reproducibility is
better than 2% rel. standard deviation.
ii) No Errors Due to Calibration
Unstability of beads suspensions over time or
counting statistics of beads cannot influence the
counting results, since no beads are used.
iii) Less Preparation or Setup Time
The sample concentration is directly analysed by
the FCM without any instrument calibration or
additional sample preparation step.
iv) Less Analysis Time
Concentration results for subpopulations, as
defined by gates, are immediately displayed on
the instrument screen. No additional analysis
steps, e.g setting gates for beads, are required.
v) Less Expenses
No reference beads required.
Performing True Volumetric Absolute Counting
Sedimentation and Count Time
Cells or other particles may tend to sediment
inside the sample tube depending on their density
in relation to the surrounding suspension solution.
Depending as well on the particle size, significant
sedimentation can take place in terms of tens of
seconds or many minutes. The CyFlow®space
analyses the concentration at the location of the
sample uptake, where it can vary over time due to
sedimentation or desedimentation effects.
Consequently, avoid too long count phase due to
too low sample speeds. Count Time typically
should not exceed 2-4 minutes.
During the count phase, user interference must be
avoided!
1. Fill 830 µl of ready prepared sample into a
sample tube (instrument setup should be set
to: prerun 2 sec, stabilize 2 sec).
2. Shake sample for resuspension of particles,
but avoid air bubbles.
3. Insert sample tube onto sample port.
- the acquisition starts and performs
prerun
stabilize
run
count.
- particles are counted during the count phase.
- the acquisition stops automatically and ready
is displayed.
4. Define subpopulations by regions and gates
to analyse concentrations
- the concentration is displayed for each
subpopulation.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 11/26
Optical Standard Setup –The Parameters
Note
If required by a specific application, the optical
standard setup can be optimized by exchanging
preassembled removable mirror/filter blocks. This
is a matter of seconds and does not require
readjustments.
Fig. 2: Examplary demonstration of the optical
bench of a CyFlow®space with 2 lasers and 8
parameters (top) and 3 lasers and 9 parameters
(bottom). Other laser light sources and parameter
allocations possible. D detector (photo multiplier)
The CyFlow®space flow cytometer can be
equipped with various laser light sources (1, 2 or 3
lasers), different laser power intensities and up to
nine optical parameters.
Due to its modular concept the optical
configuration can be adopted to many different
clinical and scientific purposes. The main
standard configurations include:
2 Laser instrument: This instrument is equipped
with a blue diode pumped solid-state Laser (20
mW) at 488 nm and a red laser diode at 638 nm
(25 mW). Up to eight optical parameters can be
included (colours reflect laser origin of the signal).
FSC: forward scatter
SSC: side scatter
FL1: green fluorescence (FITC)
FL2: orange fluorescence (PE)
FL3: red fluorescence I (PE-Cy5)
FL4: far red fluorescence I (PE-Cy7)
FL5: red fluorescence II (APC)
FL6: far red fluorescence II (APC-Cy7)
Other parameter allocations possible
3 Laser instrument, high power laser 488 nm:
This instrument is equipped with a blue diode
pumped solid-state Laser (200 mW) at 488 nm, a
violet laser diode at 405 nm (100 mW) and a red
laser diode at 638 nm (25 mW). Up to nine optical
parameters can be included (colours in the table
below reflect laser origin of the signal).
FSC: forward scatter
SSC: side scatter
FL1: green fluorescence (FITC)
FL2: orange fluorescence (PE)
FL3: red fluorescence I (PE-Cy5)
FL4: far red fluorescence I (PE-Cy7)
FL5: red fluorescence II (APC)
FL6: far red fluorescence II (APC-Cy7)
FL7: blue fluorescence (CFP, Alexa 405)
Other parameter allocations possible
Other lasers available:
532 nm green solid state laser (50 or 100 mW)
375 nm UV laser diode (16 mW)
More lasers on request
The instrument is equipped with a CCD camera in
order to monitor the particle flow and to check the
focus of the objective.
Laser
488 nm
Laser
405
nm
Laser 638 nm
Flow cell
M
M
M
D
FSC
SSC
FL3
FL1
FL4
FL5
FL2
FL6
Laser
optic
CCD
D
D
D
D
D
D
D
FL7
D
Laser
488 nm
Laser 638 nm
Flow cell
M
M
D
FSC
SSC
FL3
FL1
FL4
FL5
FL2
FL6
Laser
optic
CCD
D
D
D
D
D
D
D

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 12/26
Optical Flow Geometry
Fig. 3: Optical geometry at the flow cuvette (schematics).
Laser light is focused into the flow cell. Laser light scattered from the particles is detected in the forward
direction range (forward scatter). Side scatter and fluorescence light is collected by the objective at a right
angle.
Instrument Settings
Instrument settings are used to optimize the
CyFlow®space acquisition for the particles of
interest. The adjustments cover the gains of the
optical detectors, e.g. the photomultiplier high
voltages, the amplification mode (lin, 3 or 4
decade logarithmic), lower and upper level
thresholds and sample speeds. Instrument
settings can be once set up for a given application
and then be saved and reloaded for later use.
The following pages will describe the instrument
settings that can be made.
Please also refer to the software manual for
details on how to change the instrument settings.
Fig. 4: Multiparameter instrument settings box
sample
(hydrodynamic focusing)
laser beam
side scatter
fluorescence forward scatter
sheath fluid
waste
A

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 13/26
The Parameter Setup Dialog Box: Pulse Height, Area and Width
Opening the
Parameter Setup
Before starting an acquisition, assure the
parameters of interest are selected and set up
according to your analysis.
Click the "Parameters..." button in the
Instrument Settings box.
The Parameter Setup Dialog Box appears.
Fig. 5: The Parameter Setup Dialog Box
Note
Parameter names
cannot be changed
after an acquisition.
Parameter names
Pulse Property
Selections
Low Pass
2 Wavelengths
Signal Delay
Check if the parameter names are according to your
optical setup. Typical names are shown in the
example above. The parameter names are
displayed on the histogram axis together with a
parameter label. To change parameter names, click
into the fields and enter the names by using the
keyboard.
For each optical channel, you may analyse three
different pulse properties: 1. The pulse height, 2. the
pulse area, and 3. the pulse width. Typically, only
the pulse height is used for analysis. In case of a
DNA analysis, the pulse area of the DNA parameter
may be selected in order to discriminate cell
doublets from single cells, which show up in the
same DNA peak.
The electrical low pass filter smoothes the signals
from the detectors by averaging the signal over a
given time. This feature is only required for lamp
light sources (not realized for the CyFlow®space).
Light beams from different lasers are directed to
different positions (spots) of the flow cuvette (please
refer to page 6). Therefore, all laser derived signals
from a laser at a spot other than spot no. 1 need to
be analyzed with a time delay of 50 µsec (2-
Wavelengths Signal Delay activated, see Fig. 5)

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 14/26
Trigger
FSC, SSC, FL1, FL2, FL3 and FL4 are selected
for detection. FSC is the leading trigger
parameter.
Leading Trigger
In order to discriminate particles of interest e.g.
cells from other particles e.g. cell fragments or
nutrition particles in cell culture, a proper trigger
has to be chosen. In the CyFlow®space, any
parameter can be used as leading trigger.
Frequently, the forward scatter parameter (FSC)
or, preferently for smaller particles, the side
scatter parameter (SSC) is used to trigger on all
particles above a certain size range. However,
especially for very small particles, e.g.
microorganisms, triggering on a fluorescence
parameter can be more efficient.
Selecting a parameter as leading trigger
parameter means: Only particles that deliver a
sufficient signal above the lower level threshold on
that parameter will be acquired. Other non-
triggering parameters are aquiring signals only for
particles that generated a valid trigger signal
(above treshhold). All other particles will not be
“recognized” by the instrument. The trigger
parameter can be used for an efficient exclusion
of unwanted particles from the analysis.
Example: Assume vertebrate leukocytes in full
blood are to be analysed. Triggering on a scatter
parameter would be difficult due to the high
number of erythrocytes in the same sample.
Staining with a DNA dye (e.g. PI) and triggering
on the DNA parameter will discriminate the
leukocytes containing a nucleus from erythrocytes
without a nucleus. This works even though there
are orders of magnitutes more erythrocytes than
leukocytes in the sample.
Trigger All
The CyFlow®space offers an additional method
for triggering which allows to trigger on all
parameters.
Example: Assume a part of the particles of
interest are exclusively red and another part
exclusively green fluorescing. Triggering on the
green fluorescence would exclude the red
particles from the analysis, triggering on red would
exclude the green. If the interest is in both
subpopulations, it is required to trigger on green
and red simultaneously. This can be done with
Trigger All.
Trigger parameter(s) and mode can be selected in
the instrument settings box. Refer to the software
operating manual for details.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 15/26
PMT High Voltage, Gain, and Log Amplification
Controls
Gain
Note
Gain and lin/log
amplification mode
cannot be adjusted
after the acquisition.
Note
Be sure not to use
too high gain values.
Increasing the gain
values too much can
cause a decrease of
amplification or a
loss of signals due
to saturation effects.
Note
The gain value does
not necessarily be
equal to the PMT
high voltage nor
does it be
proportional to the
amplification.
Log
Typical effect of
change
DNA distribution of
bull sperms with X-
and Y-chromosomes.
Gain = 445.
DNA distribution of
bull sperms with X-
and Y-chromosomes.
Gain = 476.5.
EAT cells, linear
amplification
EAT cells, log 3 (3
decade) amplification
Adjustment
By means of the gain, the signal amplification can be
individually adjusted in a wide range for each
parameter. For the used photomultiplier tubes
(PMTs) by changing the gain value, the PMT's high
voltage is adjusted.
The gain value of a parameter can be selected by
clicking into the corresponding gain value field in the
instrument settings box (see Fig. 4). By using the
Right (Left) buttons gain values are increased
(decreased).
In case linear amplification is used, peaks are
expanded to the right (compressed to the left) when
increasing (decreasing) the gain (see example on
the left).
In case logarithmic amplification is used, peaks are
moved to the right (left) when increasing
(decreasing) the gain.
Use the gain to move the peaks of interest to a
suited position in the histograms.
Start with low gain values around 250. When
increasing the gain, make sure the most intense
signals still appear in the histogram and are not
moved out of scale.
Increasing (decreasing) the gain by 50
approximately causes an increase (decrease) of
amplification by a factor of two. Gain values between
250 and 600 are a good start for adjustment.
For each parameter, you can choose between linear
or logarithmic amplification. In logarithmic
amplification mode, the signal range can be set to 3
(log3) or 4 decades (log4), corresponding to a range
of 1...1000 or 0.1...1000. Generally, a logarithmic
amplification is advisable if particles with a broad
range of intensities above 1:10 are to be analysed.
Click the little arrow, select one of the following
lin (linear)
log3 (logarithmic, 3 decade range)
log4 (logarithmic, 4 decade range)
and click it.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 16/26
Sample Speed
Speed
Note
For a high accuracy
measurement, a low
speed smaller than
1.0µl/s is advisable.
Typical effect of
change
EAT cells, Speed
= 3µl/s
EAT cells, Speed
= 20µl/s
Adjustment
By means of the speed value, the sample speed (in
terms of µl/s) is set.
The speed value can be increased (decreased) by
clicking into the corresponding field in the instrument
settings box and using the Right (Left) buttons (see
Fig. 4).
The count rate increases with elevated speed
values. If the speed is increased too much, peaks in
the histograms may become wider as a result of
decreasing accuracy.
If the speed is too low, particle sedimentation effects
can influence a counting result.
Typical Speed Values
a) High accuracy measuremens
(e.g. DNA with < 1.5% CV):
Speed = 0.5 µl/s.
b) Fast measurements and absolute counting:
Speed = 4.0 - 10.0 µl/s
Fig. 6: Sample Transporting System of the CyFlow®space.

Operating Basics
Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 17/26
Threshold: Lower Level (L-L)
Lower Level (L-L)
Note
L-L and U-L cannot be
adjusted after the
acquisition.
After acquisition, you
may use 1P ranges or
2P polygons to gate out
undesired signals
instead.
Note
L-L values do not
directly correlate with
axis values.
Typical effect of
change
L-L = 10
L-L = 40
Adjustment
Lower Level L-L
Setting a proper lower level (L-L) avoids the
acquisition of small and unwanted background or
"noise" signals below a threshold.
The L-L value of a parameter can be increased
(decreased) by clicking into the corresponding L-L
value field in the instrument settings box and using
the Right (Left) buttons (see Fig. 4).
Start with a low L-L (e.g. 10). Increase the L-L until
no more of the small "noise" signals appear in the
histograms. Make sure not to remove signals from
particles of interest by a L-L being too high.
A L-L range of 0…999.9 corresponds to the full
histogram scale, independent of the channel
resolution actually selected.
If a leading trigger is selected, then the L-L can only
be effectively set on this trigger parameter.

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 18/26
Appendix
Installation Requirements
Line Power
CyFlow®space Basic Unit AC 100-240 V 50-60 Hz, 250 VA max
Instrument Size
CyFlow®space Basic Unit 560 mm x 650 mm x 300 mm (W x D x H)
Operating Environment
The CyFlow®space should be placed on a solid base, e.g. a laboratory table. It must be placed horizontally.
Reduce smoke, dust, vibrations, direct sunlight and direct neighbourhood of heatings as possible. The
installation room must be well ventilated and dry.
Temperature 15-30 C
Humidity 20-85% relative (non-condensing)
Room Clean environment. Direct sun light should be avoided.
Minimum Recommended Work Space
Table dimensions and space on desktop 120 cm x 90 cm x 80 cm (W x D x H)
Fig. 7: Recommended placement of CyFlow®space components
CyFlow
flow cytometer
Computer
Keyboard Mouse
Screen Fluid
containers
Printer
CyFlow
flow cytometer
Computer
Keyboard Mouse
Screen Fluid
containers
Printer

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 19/26
Instrument Setup –Overview
Fig. 8: Instrument Setup and periphery (CyFlow®space shown from the rearside).
CyFlow
(rearside)
Screen
Computer (rearside)
Printer
CCD Camera
Interface
Monitor
Printer
CCD
Computer
AC
AC
AC
AirSheath
Mouse
Keyboard
Sheath Waste
AC
Waste
CyFlow
(rearside)
Screen
Computer (rearside)
Printer
CCD Camera
Interface
Monitor
Printer
CCD
Computer
AC
AC
AC
AirSheath
Mouse
Keyboard
Sheath Waste
AC
Waste

Rev. 007 Date 2010-01-13 CyFlow®space Instrument Operating Manual 20/26
Instrument Setup –Step by Step
Fig. 9: Computer connection on the CyFlow®
space rearside.
Fig. 10: Power connection and laser switch on the
left side of the CyFlow® space.
Fig. 11: Tube connections for sheath and waste
containers and cable connections for the fluid
level sensors on the right side of the CyFlow®
space.
Note: Before first operation remove protection
material from inside the system!
1. Setting up the Computer
Connect printer, screen, keyboard and mouse to
the computer with the appropriate connection
cables.
Connect computer, screen and printer to AC
power line.
2. Connecting the CyFlow®space to power line
The CyFlow®space must be operated with AC
100/240 V 50/60 Hz.
3. Connecting the CyFlow®space to a
Computer
The CyFlow®space runs with a standard
Windows®PC or notebook (optional). This makes
the latest computer technology available at any
time. The integrated FloMax®software controls all
functions of the CyFlow®space, e.g. instrument
settings, sample supply, sheath pressure and
sample flow rate.
The Computer connections of the CyFlow®space
are located at the rear side of the instrument.
Connect the CyFlow®space to the computer
with the interface cable. The laser connection is
only required for the 200 mW 488 nm laser.
There is a CCD camera inside the CyFlow®space
optical bench to monitor the focus of the objective
and the sample flow inside the cuvette. Connect
the camera signal outlet (CCD) to the Video In
connection of the computer with the video
connection cable.
Network support of the CyFlow®space is provided
through a standard ethernet connection by the
Windows®computer. Please refer to the
Windows®manuals for details on how to set up a
network connection.
4. Connecting the CyFlow®space to sheath
and waste containers
Connect the sheath tube and the air tube of
the sheath fluid container to the corresponding
connectors at the right side of the CyFlow®space.
Connect the waste fluid container to the waste
outlet at the right side of the CyFlow®space.
All tubes are labeled accordingly.
Note
Make sure that the sheath and waste fluid bottles
stand on the same level as the instrument to
prevent that hydrostatic pressure is influencing the
sheath fluid pressure.