Partec CyFlow Cube 6 User manual

CyFlow®Cube 6
Instrument Operating Manual
CyFlow®Cube 6

CyFlow®Cube 8 - Instrument Operating Manual
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For in vitro diagnostic use with Partec recommended IVD reagents.
The PartecCyFlow®Cube Flow Cytometer complies with the European IVD Directive 98/79/EC and is therefore CE marked.
IVD
Contact Information:
Partec GmbH • Otto-Hahn-Straß3 32 •D-48161 Münster •Germany
Tel [phone] • Fax [phone]
E-mail: [e-mail]
© 2012Partec GmbH

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Table of content
FOREWORD ................................................................................................................... 5
PRESENTATION............................................................................................................. 6
BASICS ........................................................................................................................................................................................... 6
What is the Partec CyFlow®Cube?....................................................................................................................................... 6
What are the applications for which the CyFlow® Cube can be used?................................................................. 6
What are topics covered by this manual? ......................................................................................................................... 6
What other manuals are available?..................................................................................................................................... 6
What should I know before operating the CyFlow®Cube? ....................................................................................... 6
IN FLOW CYTOMETRY,WHAT IS................................................................................................................................................. 7
… a parameter?.............................................................................................................................................................................. 7
… a one-parameter histogram? ............................................................................................................................................. 7
… a histogram channel?............................................................................................................................................................. 7
… the count in a histogram? .................................................................................................................................................... 7
… a peak?.......................................................................................................................................................................................... 7
… background in a histogram?............................................................................................................................................... 7
Example of a histogram............................................................................................................................................................. 8
Example of a dotplot ................................................................................................................................................................... 8
Histogram and dotplot in immunology.............................................................................................................................. 9
INSTRUMENT STARTING PROCEDURE.....................................................................................................................................10
Sheath fluid level control and/or refill .............................................................................................................................10
MAIN CYVIEW™LOGIN WINDOW.............................................................................................................................................11
USER LEVELS...............................................................................................................................................................................12
CYVIEW™MAIN PAGE –AFTER SUCCESSFUL LOG-IN..........................................................................................................13
CYVIEW™CONTROLS................................................................................................................................................................13
1D/2D PLOTS–display options............................................................................................................................................14
RESULTS .........................................................................................................................................................................................15
REGIONS .........................................................................................................................................................................................16
GENERAL - properties of the instrument.........................................................................................................................16
ACQUISITION –instrument control ...................................................................................................................................17
MEASURE –definition of measure modes .......................................................................................................................17
Measure modes ............................................................................................................................................................................18
Analyze all (default selection) ..............................................................................................................................................18
Volumetric Counting with Volume .....................................................................................................................................18
Volumetric Counting with Electrodes ...............................................................................................................................19
Cells in Region ..............................................................................................................................................................................19
CYVIEW®INSTRUMENT/MEASUREMENT SETTINGS.........................................................................................................20
PRIME/WORK/CLEAN .............................................................................................................................................................20
Priming / Initialization process...........................................................................................................................................20
Work process ................................................................................................................................................................................22
Shut down process......................................................................................................................................................................22
INTERMEDIATE CLEANING PROCESS .......................................................................................................................................23
SHEATH AND WASTE BOTTLE..................................................................................................................................................23
MEASUREMENT PARAMETERS.................................................................................................................................................24

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Trigger properties (P) ..............................................................................................................................................................24
Threshold settings (T) ..............................................................................................................................................................24
Flow speed (S) ..............................................................................................................................................................................24
Light source (L) ...........................................................................................................................................................................24
1D/2D PLOT PROPERTIES.......................................................................................................................................................25
Plot name........................................................................................................................................................................................25
X Log On/Y Log On.....................................................................................................................................................................25
Erosion levels................................................................................................................................................................................25
X/Y Channel...................................................................................................................................................................................25
BitRange .........................................................................................................................................................................................25
CR-Mode..........................................................................................................................................................................................25
REGION/ROI PROPERTIES .......................................................................................................................................................26
Create a polygonal region ......................................................................................................................................................26
Region properties .......................................................................................................................................................................26
Region name .................................................................................................................................................................................26
Home Plot name..........................................................................................................................................................................26
Color RGB........................................................................................................................................................................................26
Max count.......................................................................................................................................................................................26
Sorter region.................................................................................................................................................................................26
Moving of regions within a histogram..............................................................................................................................26
Create a vertical or horizontal histogram splitter......................................................................................................27
Create a quadrant assembly..................................................................................................................................................27
Change layout for regions.......................................................................................................................................................27
Applying regions to other plots  Gating Function...................................................................................................27
COMPENSATION .........................................................................................................................................................................28
TYPICAL SAMPLE ANALYSIS.....................................................................................................................................................29
KEYBOARD/MOUSE COMBINATIONS....................................................................... 31
APPENDIX .................................................................................................................... 32
BIOHAZARDS...............................................................................................................................................................................32
MAINTENANCE ...........................................................................................................................................................................33
Service..............................................................................................................................................................................................33
Transport and Storage.............................................................................................................................................................34
Disposal ...........................................................................................................................................................................................34
LASER SAFETY............................................................................................................................................................................35
TECHNICAL SPECIFICATIONS ...................................................................................................................................................36
Optical Standard Setup ........................................................................................................................................................36
NOTES .......................................................................................................................... 39

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Foreword
The CyFlow®Cube Instrument Operating Manual is aimed for a large spectrum of users, from beginner up
to the most skilled flower. The beginner or casual user will find the key functions and concepts to use the
Cube and its software. The confirmed flower will find an in-depth detail of the inner working and parameters
of the Cube to customize its use and obtain optimal performances.
CyView™for Cube 6 is the instrument operation software for the CyFlow®Cube 6 (CY-S-3060). Partec is
continuously working on CyView™to better fulfill your demands. If you have questions concerning this
manual or the software, find problems associated with CyView™or you have a good suggestion to be
included in a new version, please let us know by sending an email or a note to Partec GmbH.
The present manual is valid as of CyView™ software version 1.5.
For more details about the reagent kits, suitable for use with the CyFlow®Cube,please refer to the
respective product data sheets. There are also several Application Notes available.
If you have questions, please contact your local distributor, one of the Partec subsidiaries, or Partec in
Germany ([e-mail]).
Further details and addresses can be found on our website at:
www.partec.com/distributors
Please do not forget to add in your request the following information:
Serial number (serial No.) of the CyFlow®Cube
Your complete contact address
This manual contains references to names and products from Partec and other companies which
are registered trademarks or protected by copyright.
Partec GmbH, CyFlow®Cube6, CyView™for Cube6, Robby®.
Microsoft®Corp.: Windows, Word, Excel, PowerPoint, Paint.
Hewlett Packard®: Deskjet Laserjet.

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Presentation
Basics
What is the Partec CyFlow®Cube?
The PartecCyFlow®Cube is a fully equipped desktop Flow Cytometer (FCM). CyFlow®Cube features a
modular optical concept. This allows using different lasers as light sources and the detection of up to 8
(CyFlow®Cube 8) or of up to 6 (CyFlow®Cube 6) optical channels (parameters). The CyFlow®Cube allows
easy optimization of the optics for any application by simple exchange of optical filters and mirrors. The
CyFlow®Cube runs with an internal PC. Data acquisition, instrument control, and data analysis are
controlled and performed by the CyView™software.
What are the applications for which the CyFlow® Cube can be used?
Together with the software, the CyFlow®Cube offers automation for routine use and flexibility for research
use for practically any flow cytometric application. The applications cover:
Routine multi-color-immuno-phenotyping
Blood Cell Analysis/HIV monitoring (e.g. CD4 cell count)
Leukocyte Counting/Rare Event Analysis
Microorganism Analysis
Fermentation Control
Particle Concentration Analysis
True Volumetric Absolute Counting
Particle Size and Fluorescence Distribution Analysis
What are topics covered by this manual?
The CyFlow®Cube Instrument Operating Manual covers the basic operation and maintenance of the
CyFlow®Cube instrument. This manual also covers details related to the software.
What other manuals are available?
Application Notes and Service Manuals are available to get started. They contain hints to achieve the
best results.
What should I know before operating the CyFlow®Cube?
This manual assumes that you have basic knowledge on 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).

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In flow cytometry, what is...
… a parameter?
In flow cytometry, parameter denotes a measured property of the
particles. Frequently, a parameter is synonymous to an optical channel.
E.g. an instrument with 6 parameters is equipped with 6 optical
detectors.
… a one-parameter histogram?
A one- parameter histogram displays the distribution of cells among a specific
property, e.g. how many cells contain a given quantity of DNA or bind a given
number of antibody molecules.
… a histogram channel?
The measured signal intensity is assigned to one of 65536 (16bit) quantity classes or channels. In a one-
parameter histogram the channels are represented on the x-axis.
… the count in a histogram?
The number of cells being assigned to a given channel is referred to as channel content or simply count. In
a one-parameter histogram, the count is shown on the y-axis.
… a peak?
All cells having about equal characteristics among the analysed cell property (e.g. content of a specific
constituent like DNA) form a peak. In the case of a typical DNA histogram one peak represents the G1 and
another peak (with twice the channel value) represents the G2/M phase of the cell cycle.
In case of immunolabelled cells often one peak for unlabelled (negative) and one peak for labelled
(positive) cells can be detected. Peaks can be analysed by identifying them with region markers.
… background in a histogram?
Histograms sometimes show undesired signals in the lower channels, frequently called ´noise´ or
´background´. These signals may originate from cell fragments or other particles resulting from sample
preparation. In case of high signal amplification, background can also be caused by particle contaminated
sheath fluid.
The lower level (L-L) or threshold?
The lower level (L-L) threshold is a mean to suppress background signals. Signals below the lower level are
rejected from the signal acquisition. To exclude noise from a histogram already acquired, a region-gate can
be used.

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Peaks
Number of events
Threshold
of lower
limit
Linear representation of SSC
Log representation
for fluorescence
Example of a histogram
A histogram represents a distribution of measured signals (events) over 1
dimension. Data can be presented on the dimensions of relative particle size or
optical particle structure (Forward Scatter (FCS) or Side Scatter (SSC)), resp.
or on their relative fluorescence intensities in different light colors (fluorescence
parameters FL1 to FL6).
In this example, the dimension represented is the relative size (FSC) on a
logarithmic scale in X, and the number of events on a linear scale in Y. Two
peaks are visible.
Example of a dotplot
A 2D dotplot presents correlated data over 2 dimensions. In the image on the right a sample of leukocytes
(after lyse of the red blood cells) is plotted with their relative light scattering (SSC) property against the
intensity of the CD45 antigen.
The Z value represents the number of events that have the same coordinates. 1 event will be represented
by a black point; if 10 events have the same coordinates, the point representing them will be grey. It will be
yellow if more than 20 points are overlaying one another. The Z scale is dynamic and will adapt during the
measurement to a scale of 1 (black) to the maximum overlaying event coordinate color coded in red.

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G
M
L
Histogram and dotplot in immunology
Lysed blood 2D dotplot
Lysed blood is represented in a dot plot presenting the FSC in X axis and SSC in Y axis. Both axes are in a
linear scale.
Three distinct groups are visible; they represent the granulocytes (G), monocytes (M) and lymphocytes (L).
Histogram and dotplot of immunological staining
This histogram represents the spectrum of the cells presented in the
previous dot plot stained with antibodies anti-CD45 conjugated to
PhycoErytrin (PE) Dy647. The X axis displays the fluorescence in a 4-dec
logarithmic scale and the Y axis displays the number of events in a linear
scale.
This dot plot presents the cells fluorescence in X on a logarithmic scale
(CD45 PE-Dy647) versus SSC in Y on a linear scale.
This data display allows an easier interpretation of 2 parametric data
compared to the histogram. Concluded from this example, the lymphocytes
are strongly stained with the anti-CD45 antibody, the monocytes are slightly
stained and the population of granulocytes is negative.

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Rear side of CyFlow®Cube showing the
main power switch and the sheath fluid and
waste bottle compartment
Instrument starting procedure
Sheath fluid level control and/or refill
Before switching on the machine, it is recommended to check the levels of the sheath fluid and waste
bottles. They can be found at the back-left of the apparatus in a sliding compartment.
Make sure SHEATH bottle is filled with 700 ml of clean, filtered, and
degassed sheath fluid and is closed with the screw top.
Please notice: Higher level of sheath fluid could lead to unstable
sample flow.
In order to guarantee highest quality of the measurements we highly
recommend use Partec Sheath Fluid (order no. 04-4007).
It is recommended to replace the sheath fluid at least once a week or
before any daily use.
When filling up the sheath fluid bottle make sure no air bubbles are
trapped in the yellow filter unit inside the bottle!
Make sure WASTE bottle is empty and the screw top is tightly
closed.
The waste bottle must be emptied after and before each user
session. When using bio-hazardous samples, a volume of 50 ml of
hypochlorite 0.5% (Order No 04-4012) should be introduce into the
empty waste bottle for initial disinfection.
Switching on the CyFlow®Cube
The power supply switch is found at the back of the Cube next to the main supply cable. The Cube is, in
default, set in a stand-by mode. The full activation of the Cube requires pressing button on the top of the
machine. The display screen must be first lift up to access it.
This will start the embedded computer, automatically start the CyView™software and load the last
employed configuration.
Casual/medium expertise user:
Once the Cube started no further steps are
necessary. You can directly start your
measurements!