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Partec CyFlow Cube Series Скачать руководство пользователя страница 7

CyFlow

®

Cube Instrument Operating Manual

Rev018_2012-01-27

7/36

In flow cytometry, what is ...

… a parameter?

… a one-parameter histogram?

… a histogram channel?

The measured signal intensity is assigned to one of 65536 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 a of 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.

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

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.

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Содержание CyFlow Cube Series

Страница 1: ...CyFlow Cube Instrument Operating Manual CyFlow Cube 6 CyFlow Cube 8 CyFlow Cube 8 Sorter ...

Страница 2: ...Partec CyFlow Cube Flow Cytometer complies with the European IVD Directive 98 79 EC and is therefore CE marked IVD For in vitro diagnostic use with Partec recommended IVD reagents Contact Information Partec GmbH Am Flugplatz 13 D 02828 Görlitz Germany Tel 49 3581 8746 0 Fax 49 3581 8746 70 E mail service partec com 2012 Partec GmbH ...

Страница 3: ...xample of a histogram 9 Example of a dotplot 9 Histogram and dotplot in immunology 10 GETTING STARTED 10 INSTRUMENT STARTING PROCEDURE 11 Sheath fluid level control and or refill 11 Switching on the CyFlow Cube 11 MAIN CYVIEW LOGIN WINDOW 12 USER LEVELS 12 CYVIEW MAIN PAGE 14 Presentation 14 CyView Controls 15 CHANNELS parameter definition 15 PLOTS display options 15 PROCCESS instrument control 17...

Страница 4: ... count 24 Sorter region 24 Colour Gating On 24 Moving of regions within a histogram 24 Create a polygonal region 24 Region properties 24 Create a vertical histogram splitter 25 Create a vertical dotplot splitter 25 Create a horizontal dotplot splitter 25 Create a quadrant assembly 25 Change layout for regions 25 Applying regions to other plots Gating Function 26 Keyboard Mouse combinations 26 APPE...

Страница 5: ...lems 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 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...

Страница 6: ...ic application The applications cover Routine multi colour 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 ...

Страница 7: ...content of a specific constituent like DNA form a peak In the case a of 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...

Страница 8: ...m 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...

Страница 9: ...nt if 10 events have the same coordinates the point representing them will be grey It will be red 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 colour coded in red A histogram represents a distribution of measured signals events over 1 dimension Data can be presented on...

Страница 10: ...ugated to PhycoErytrin PE 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 FITC 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 th...

Страница 11: ...mmended 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 800 ml of clean filtered and degassed sheath fluid and is closed with the screw top In order to guarantee highest quality of the measurements we highly recommend use Partec Sheath Fluid order no 04 4007 It is recommend...

Страница 12: ...ive Password Cube1 case sensitive Login as a standard USER The code will be given to you by the main user s of the instrument The main user has the rights to define or delete users During start of the instrument the automated self testing procedures are processed XML configuration displays the correct loading of the setting files Searching Device display of the correct recognition of the connectio...

Страница 13: ...ame and Password followed by Build new account Main user should be deactivated As User the own password can be changed by selecting the Name and placing a new Password followed by Change my password As Main User the own password can be changed by selecting the Name and placing a new Password followed by Change my password As Main User any User account can be deleted by selecting the Name followed ...

Страница 14: ...egion statistics Results table Instrument settings L M Measuremen t interface and V D Buttons allowing to load and save data files fcs data files Buttons dedicated to saving and retrieving instrument settings xml data files or configuration scripts Button for the generation of a PDF report of the acquired data Console displaying the control board status Status of sample high counting phase empty a...

Страница 15: ...n the used Configuration Script Plots are named H1 Hx for histograms and P1 Px for dotplots As specific plot is selected with the arrow keys Define a Comment characterizing the plot e g FSC Switch between Lin and Log scale for X and Y Axis Define an Erosion level for dot to be displayed z axis level e g an Erosion level of 2 shows only dots representing 3 and more signals Select X axis channel and...

Страница 16: ...o the formula Counter results on refers to the result of a volumetric counting Confirm all modifications by pressing Accept Home Plot Name defines the plot the region refers to Color RGB defines the region s color and its color in color gating Max Count defines a maximum count for an Events in Region particle limit Sorter Region On activates region as sorter region only in CyFlow Cube Sorter Color...

Страница 17: ...Defines sample port electrode Volume in µl µlPerSec mBar defines a sheath fluid flow parameter SW Version specifies current software version Mask or Positive selects the trigger parameters multiple trigger parameters are logical or connections Voltage of the individual optical parameters 0 volt 999 volts defines PMT signal amplification Threshold defines the trigger signals cut off level on 4 dec ...

Страница 18: ...tric Counting with Volume Events in Region Speed values and Volume values can be edited and stored in FCS file and Config script Confirm all modifications by pressing Accept SCRIPT definition of measure modes Schematic overwiew of the measure modes with A flash indicates clearing of the data User selectable criteria in green software defined values in red ...

Страница 19: ... stabilization of sample flow Start Measure Data acquisition Console displaying control board status During the data acquisition the total particle count and the analysis rate is indicated as a sliding bar During the acquisition phase it is possible to manipulate the instruments set up e g by changing samples speed PMT voltage and threshold levels Use CLEAR button to erase data after manipulations...

Страница 20: ...olumetric counting with volume the counting volume is flexible and can be pre selected by the user In a first analysis phase the sample is acquired normally as in continuous acquisition mode Reaching the pre selected volume the data are cleared and the volumetric counting phase starts and the pre selected volume will be analyzed The volume can be used as the basis for concentration determination E...

Страница 21: ...ck clean Then the same operation must be repeated with distilled water The Cube can be put in standby by closing CyView Menu Exit and closing WindowsXP start Exit Switch Computer Off To power off completely the Cube use the main power switch at the back panel The waste bottle content must be discarded accordingly to the relevant biohazard regulations A regular thorough cleaning of the sheath fluid...

Страница 22: ...ight source L This option allows to switch on off the light sources Trigger properties P Single trigger The trigger is the parameter defining if a signal gets recorded Only if the trigger parameter detects a signal other parameters of the system will record signals In other words if a trigger is set in FSC to record only bigger particles e g threshold set at 0 3871V with a gain value of 170V e g o...

Страница 23: ...oints will not be displayed allowing a better visual discrimination of the higher frequency data signal against background X Y Channel The displayed parameters can be chosen from the drop down menu giving the user the list of the activated parameters Mode Graphical model for the data line histogram filled area histogram overlay BitRange The values are ranging from 6 to 12 It sets the channel resol...

Страница 24: ...lot selected as colour gating only illustrated in the graph on the left hand side with an overlay of the staining of lymphocytes in red overlaying the blue histogram presenting all events Max count This option allows to fix the maximum number of events from a particular ROI Region Of Interest to be analysed see CyView 8 SCRIPT register Sorter region To set the ROI region as a sorting gate just by ...

Страница 25: ...r into the dotplot and press the right mouse button Select Built horizontal splitter region to divide the dotplot into two sections HS1 1 and HS1 2 The intersection can be modified by moving the cursor into the dotplot and keeping the left mouse button pressed Create a quadrant assembly To create a quadrant assembly within a dotplot move the cursor into the dotplot and press the right mouse button...

Страница 26: ... button outside a region area allows to select any existing region which is labeled with Color gating on Display options are Show all events shows all events in pseudo 3D color Show color regions only shows the events in the selected region s in pseudo 3D color Show colors only shows the events in the selected region s in the region color All Region colors shows the events in the selected region s...

Страница 27: ... steps click and hold left mouse Ctrl Moves a region in small steps Region click and hold left mouse Shift Moves a region to the original position click and hold left mouse Shift Ctrl Moves the region by addition of an interval to the original position in very small steps click and hold left mouse M Move the regions name over 5 positions N E S W central Right mouse Alt Opens mouse menu Axis of a p...

Страница 28: ... international as well as national regulatory standards such as GLP good laboratory practice must be met for all users Therefore the system is marked with the following biohazard label Warning The Waste may contain biohazardous and carcinogenic material from the samples infectious material dyes Warning biohazards ...

Страница 29: ...d clean with warm detergent solution and a brush Clean sheath reservoir with distilled water and a clean brush and flush with clean distilled water several times Remember a clean sheath fluid reservoir is critical for proper operation If the CyFlow Cube will not be used for longer periods clean flow system by using distilled water Put a sample tube half ways filled with distilled water at the samp...

Страница 30: ...nsport or storage please take care that the system will be stored under the following conditions Temperature 5 50 C Humidity 20 85 relative non condensing Room Clean environment no direct sun light Disposal In case of product disposal please proceed according to the Partec standard operating procedure SOP for decontamination After decontamination the system has to be disposed according to the loca...

Страница 31: ...tted if the housing of the device is damaged and the protection cover for the laser beam is removed Therefore the system is marked with the following laser safety labels Warning It is prohibited to open the instrument as it is equipped with a class 3b laser unit Warning laser radiation Attention Laser radiation Class IIIb if cover is removed and shutter is opened Additional explanation ...

Страница 32: ...scence fluorescence origin 638 nm laser APC 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 any re adjustment The CyFlow Cube flow cytometer can be equipped with various light sources and up to eight optical parameters Depending on the number of light sources...

Страница 33: ...unting per volume True Volumetric Based on precise counting and mechanical fluid volume measurement Absolute Counting No need for reference sample or beads Instrument Check Partec Count Check Beads Partec Calibration Beads 1 µm and 3 µm Partec DNA Control PI Set up Time Max 5 minutes Parameters Up to 8 optical parameters FSC SSC FL1 FL2 FL3 FL4 FL5 FL6 Particle Size Range 0 1 µm 50 µm standard cuv...

Страница 34: ...liptical 15 µm x 100 µm at 488 nm Other beam geometries upon request 3 CyFlow Cube Fluidics Flow Cuvette Synthetic quartz flow cuvette 350x 200 µm for laminar sample transport with sheath fluid fluorescence forward and side scatter light detection Sample Delivery Computer controlled precision syringe pump for contamination free sample transport Built in vacuum pump for waste container Vacuum press...

Страница 35: ...CyFlow Cube Instrument Operating Manual Rev018_2012 01 27 35 36 Notes ...

Страница 36: ...CyFlow Cube Instrument Operating Manual Rev018_2012 01 27 36 36 Excellence in Flow Cytometry and Cell Analysis ...

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