ChemoMetec NucleoCounter User Manual
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ChemoMetec NucleoCounter User Manual

Mammalian cell counter
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NucleoCounter
®
User's Guide
P/N 991-0009
Revision 1.4
Technology that counts

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Summary of Contents for ChemoMetec NucleoCounter

  • Page 1 NucleoCounter ® User's Guide P/N 991-0009 Revision 1.4 Technology that counts...
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  • Page 3  NucleoCounter Mammalian Cell Counter P / N 9 9 1 - 0 0 0 9 ( E n g l i s h ) R e v i s i o n 1 . 3 J u n e , 2 0 1 5 ChemoMetec A/S Gydevang 43, DK-3450 Allerød, Denmark...
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  • Page 5 ChemoMetec A/S assumes no liability resulting from errors or omissions in this document, or from the use of the information contained herein. ChemoMetec A/S reserves the right to make changes in the product design without reservation and without notification to its users. Copyright Notices Copyright ©...
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  • Page 7 Declaration of Conformity  Name of product: NucleoCounter Type: Mammalian Cells Other identifying data: Part no. 900-0004 The product complies with requirements of the following directives: 89/336/EEC - Electromagnetic Compatibility (EMC) 89/392/EEC - Machinery Directive Harmonized standards, which have been used: EN61326: 1997+A1: 1998 (Class B) +A2: 2000, Electrical equipment for measurement, control and laboratory use –...
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  • Page 9: Weee Directive Information - Europe Only

    WEEE directive information – Europe only Correct Disposal of This Product (Waste Electrical & Electronic Equipment) - Europe only This marking shown on the product or its literature, indicates that it should not be disposed together with other household wastes at the end of its working life.
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  • Page 11: Introduction And Intended Use

    The NucleoCounter type at ChemoMetec A/S is 900-0004. This user´s guide is valid for NucleoCounter software version 4.XX The NucleoCounter system enables the user to obtain cell counts and viability determinations on cell suspensions from a wide range of cultured mammalian cell lines.
  • Page 12 The NucleoCounter, NucleoCassette™, the Lysis buffer and the Stabilizing buffer are not intended for human or veterinary consumption. The NucleoCounter system is for research use only, not for diagnostic use.
  • Page 13 Introduction and intended use This page was intentionally left blank...

  • Page 14: Warnings And Precautions

    Power and cables Use the shielded USB cable supplied with the NucleoCounter to ensure that you maintain the appropriate EMI classification for the intended environment. The USB interface connector of the NucleoCounter must only be connected to SELV circuits.
  • Page 15 E nclosure Figure 1 Printer Output connector seen from the cable entry side. NucleoCounter is powered by an external 12VDC power supply. For safe use, please follow the instructions for connecting the power supply. The NucleoCounter shall only be used with one of the following external power supplies: ...
  • Page 16 The mains supply cord and plug of the external power supply shall comply with any national regulations. The user shall be made aware of that, if the NucleoCounter and the external power supply is used in a manner not specified by the manufacturer, the protection provided by the NucleoCounter and the external power supply may be impaired.
  • Page 17 Warnings and precautions Cassettes, Reagents and Dispensers With respect to usage and handling of cassettes, lysis buffers and dispensers, please refer to appropriate package inserts for these items. Caution! When using a bottle-top dispenser: To protect against accidental splashes protective clothing, eye protection and gloves must be worn when using potentially hazardous liquids.

  • Page 18: Table Of Contents

    4.1.1 Starting up ........14 4.1.2 Shutting down ........15 ..............15 NSTALLATION OF UCLEO OPERATION OF THE NUCLEOCOUNTER ............16 ..................... 16 EADY MODE ............. 16 NSERTING AND REMOVING THE CASSETTE 5.2.1 Protection of the optical system ....16 5.2.2 Inserting cassette .........
  • Page 19 OUNTING NON VIABLE CELLS ................. 29 ALCULATE VIABILITY ................. 30 ILUTE THE SAMPLES MAINTENANCE OF NUCLEOCOUNTER ............33 ....................33 LEANING 7.1.1 Cassette insertion area ......33 7.1.2 Optical elements ........33 7.1.3 Removal of dust particles ......34 7.1.4 Spill of liquid ........35 TROUBLESHOOTING - ERROR MESSAGES ..........

  • Page 20: Inspection And Unpacking Of Equipment

    1 Inspection and Unpacking of Equipment 1 Inspection and Unpacking of Equipment Upon receiving the order from ChemoMetec A/S, the box or boxes should be carefully inspected for any damage that may have occurred during shipping. Any damage must be reported to the carrier and to ChemoMetec A/S immediately.
  • Page 21 1 Inspection and Unpacking of Equipment This page was intentionally left blank...

  • Page 22: Introducing The Nucleocounter

    2 Introducing the NucleoCounter 2 Introducing the NucleoCounter The NucleoCounter method provides a fast alternative to the manual techniques, yielding objective and reliable results. Furthermore the NucleoCounter technology provides improved safety to the user compared to other comparable methods. The NucleoCounter system is comprised of the NucleoCassette for...

  • Page 23: Fluorescence Microscope

    Figure 3. Figure 3. The NucleoCounter´s compact fluorescence microscope Before the NucleoCounter fluorescence microscope is activated a NucleoCassette is loaded with sample and inserted into the insertion slit of the instrument. The lid is closed in order to exclude external light from interfering with the analysis.

  • Page 24: Built-In Image Analysis

    DNA contained in the measurement chamber of a NucleoCassette 2.1.2 Built-in image analysis The NucleoCounter is equipped with advanced software for image analysis. This integral software analyzes the recorded image and counts the number of nuclei in the image. As the volume of...

  • Page 25: The Nucleocassette

    The entire process takes approximately 30 seconds, from pressing “Run” to the presentation of the result in the display of the NucleoCounter. Performing a cell count with the NucleoCounter is objective and the results are independent of the operator. The NucleoCassette The NucleoCassette is a custom made disposable plastic device designed for optimal sample handling and safe disposal.

  • Page 26: Sample Volume

    Therefore, the area is constant and specific for each instrument. 2.2.4 Cassette handling The built-in image analysis method of the NucleoCounter offers considerable stability, when eliminating non-cellular objects from the image, including scratches and smears on the cassette windows, and thus producing valid results even under extreme conditions.
  • Page 27 2 Introducing the NucleoCounter propidium iodide. Furthermore, when using the Lysis buffer, the cell aggregates are dissolved. The main objective of the Stabilizing buffer is to raise pH of the sample mixture, thereby optimizing the fluorescence of propidium iodide and to stabilize the cell nuclei.
  • Page 28 Fejl! Henvisningskilde ikke fundet. This page was intentionally left blank...

  • Page 29: Control Buttons, Keypad And Display

    3 Control Buttons, Keypad and Display Interactive Controls 3.1.1 Control Buttons There are four Control Buttons on the front of the instrument (see Figure 6): Control Buttons on NucleoCounter Table 3. Use this button to turn the instrument On/Off on and off...

  • Page 30: Keypad

    3 Control Buttons, Keypad and Display 3.1.2 Keypad The Keypad is comprised of 12 keys (see Figure 6): “0-9”, “F” (function key) and “” (Enter). The keypad is used for any entering of values used to alter the status of the instrument. Interface Display 3.2.1 Status indicator A small Status Indicator light above the “On/Off”...

  • Page 31: Installation And Start-Up

    Figure 7. Connecting the 12VDC power cord plug to the NucleoCounter The NucleoCounter can now be turned on and off by pressing the “On/Off” button on the front panel. 4.1.1 Starting up Caution! During start-up the NucleoCounter performs a self-check.

  • Page 32: Shutting Down

    4 Installation and Start-up The start-up display shows the version number and date (year- month-date) of the internal software of the NucleoCounter in line 3 (“v4.XX” in Figure 8). The serial number of the instrument is shown in line 4 in the display (“002-01” in Figure 8).

  • Page 33: Operation Of The Nucleocounter

    Unless inserting/removing a cassette or cleaning the inserting area or the lid, the lid should be kept closed at all times as means of protecting the optical system of the NucleoCounter. 5.2.2 Inserting cassette To insert the cassette: 1.

  • Page 34: Measuring - The Run Button

    5 Operation of the NucleoCounter 4. Close the lid gently. After analysis the lid is lifted and the cassette is removed from the insertion slit and disposed of according to national or regional laws or regulations regarding the nature of the mixture it contains.

  • Page 35: Result Presentation

    Cell density of the cell lyzate, presented in Result mode 3. 5.4.1 Result mode The NucleoCounter allows the results to be presented using one of three different representations of the results. In the default display mode (mode 3) the result is displayed with number of digits corresponding to full representation of the number of cells per µl (with 1, 2, 3 or 4 digits).

  • Page 36: Saving Parameters

    To change the representation of the results follow the directions below (a letter or number enclosed with "<" and ">" corresponds to a button on the keyboard).  Press <F> <2> <1> on the keyboard of the NucleoCounter.  Press enter <>. ...

  • Page 37: Setting Date (F200) And Time (F201)

    5 Operation of the NucleoCounter 5.4.3 Setting Date (F200) and Time (F201) The functions, F200 (“F + 2 + 0 + 0 + ”) and F201 (“F + 2 + 0 + 1 + ”), are used to set the time and the date, respectively.

  • Page 38: Lcd Contrast (F220)

    If the value has been changed the new contrast will be remembered during the session. When shutting down the NucleoCounter asks if the new value shall be saved as the new default value. Press “” to save or “Esc” if the present default value should not be overwritten.

  • Page 39: Zero Count Check (F50)

    5 Operation of the NucleoCounter Figure 17. Instrument Info The display shows the name of the instrument in line 2 of the display. The version number and date (year-month-date) of the integral software of the instrument is shown in line 3. The serial number of the instrument is shown in line 4 of the display.
  • Page 40 Figure 23 The Zero Count Check did not pass the test If the Zero Count Check returns an error, it is recommended to lean the insertion slit, e.g. with compressed, dry air (refer to chapter 7 Maintenance of NucleoCounter and Technical Note...
  • Page 41 5 Operation of the NucleoCounter No. 004 “How to clean the NucleoCounter”). Perform the Zero Count Check again. If the error appears even after several attempts to clean the insertion slit with compressed air and if  necessary the NucleoCounter...
  • Page 42 5 Operation of the NucleoCounter This page was intentionally left blank...

  • Page 43: Total And Viability Count

    Furthermore various alternatives for diluting the samples are presented. The fundamentals on which the NucleoCounter is based are discussed thoroughly in chapter 2 over. The NucleoCounter method is based on staining mammalian cell DNA with a fluorescent dye, propidium iodide.

  • Page 44: Total Cell Count

    6 Total and viability count  Pipette and tips  Dispensettes if convenient  Sample vials (approx. 1,5 ml) Remember- samples obtained from the cell suspension must be representative. Total cell count To determine the total concentration of cells draw a sample of the cell suspension into a vial.
  • Page 45 Figure 24 Load the NucleoCassette by immersing the tip in the stabilized lyzate and gently press the piston. Open the lid of NucleoCounter and insert the loaded NucleoCassette. Then close the lid and press “Run”. The lid has to be closed during the analysis to eliminate any external light from the NucleoCassette chamber.

  • Page 46: Counting Non-Viable Cells

    Perform the measurement (for details see sections 5.2 and 5.3). The result is displayed on the NucleoCounter and the computer if one is connected after approximately 30 seconds. Examine the result to make sure that the concentration of cells is in the measurement range 5•10...

  • Page 47: Dilute The Samples

    100% 98,5%    Dilute the samples As mentioned earlier the NucleoCounter is able to count cells in the range 5x10 - 2x10 cells/ml, the optimal range being 1x10 – 2x10 cells/ml. Therefore it can be necessary to dilute or concentrate the cell samples.
  • Page 48 The multiplication factor is calculated from the two separate dilutions. In this example the multiplication factor is 5x3 = 15. Therefore the concentration of cells obtained from the NucleoCounter has to be multiplied by 15 to calculate the concentration of cells in the cell suspension.
  • Page 49 6 Total and viability count This page was intentionally left blank...

  • Page 50: Maintenance Of Nucleocounter

    When cleaning the cassette insertion area, great care must be taken against introducing any liquid or dust into the insertion slit of the NucleoCounter. Any liquid that enters the interior of the NucleoCounter can da mage the optical parts and thus compromise the quality of the cell counts.

  • Page 51: Removal Of Dust Particles

    7.1.3 Removal of dust particles The presence of a contaminant (e.g. dust particles) will normally not influence the counting of cells. The NucleoCounter will distinguish between cells and contaminants, since cell is generally significantly smaller than contaminants.

  • Page 52: Spill Of Liquid

    Even though such phenomena are clearly visible on the image it only rarely affects the results of the NucleoCounter. Since it is possible to damage the optical system while attempting to clean the system it is recommended that ChemoMetec A/S should...
  • Page 53 7 Maintenance of NucleoCounter This page was intentionally left blank...

  • Page 54: Troubleshooting - Error Messages

    8 Troubleshooting - Error messages Under certain conditions the NucleoCounter will display error messages during operation. Corrective actions are suggested below but if they do not correct the errors, contact ChemoMetec A/S or the local distributor. No valid cassette When trying to analyze a cassette, which is not valid, the NucleoCounter displays the error message shown in Figure 27.

  • Page 55: Actuator Error Messages

    If the cassette was inserted properly turn the NucleoCounter off and on a few times. If the actuator moves during start-up it is likely that the error has been corrected.

  • Page 56: Sensor Error

    Sensor error The black dots, which are printed on the cassettes are read by sensors inside the NucleoCounter each time an analysis is run. The dots code for the volume of lyzate mixture in the cassette chamber. If one or more of the sensors has a faulty operation an error message will be displayed, see Figure 31.
  • Page 57 Figure 32. Inspect the fuse. If it needs replacement use a fuse: 1.25A/250Vac (UL 248). If the NucleoCounter does not turn on after replacement of the fuse, or if the fuse was not broken, please contact ChemoMetec A/S or the local distributor for further instructions.
  • Page 58 8 Troubleshooting - Error messages This page was intentionally left blank...

  • Page 59: Technical Specifications

    65 percent relative humidity at maximum 35°C; minimum temperature 15°C. USB, version 1.1. Note: Does not support USB Hubs. Thermal or Impact printer with RS-232 Printer interface and cable supplied by ChemoMetec A/S. Other environmental operating conditions for the NucleoCounter Indoor use only.

  • Page 60: The Nucleocassette

    See documentation delivered with the Stability NucleoCassettes. EMC/EMI standards The NucleoCounter complies with EMC/EMI standards as follows. EN61326: 1997+A1: 1998 (Class B) +A2: 2000, Electrical equipment for measurement, control and laboratory use – EMC requirements (emission and immunity). Annex B and C from A1: 1998 is used.

  • Page 61: Equipment And Accessories

    10 Equipment and Accessories 10 Equipment and Accessories 10.1 Equipment and Accessories List The equipment and accessories for the NucleoCounter concept are listed in the table below. Ensure that these are the only equipment and accessories used together with the NucleoCounter.

  • Page 62: Power Supply

    NucleoCounter 10.2 Power supply The NucleoCounter shall only be used with one of the following external power supplies:  Power supply Class I, Proton Electronic Industrial Co. Ltd., model SPN-260-12, input rated 100-240 Vac, 50-...

  • Page 63: Mains Power Cord

    The detachable power supply cord set and appliance inlet of the external power supply are considered as the disconnecting device. Contact ChemoMetec A/S for information on order of a specific power supply listed above. 10.3 Mains Power cord Detachable power supply cord set for the Class I specified...

  • Page 64: Dry Compressed Air

    The Mains supply cord and plug of the external power supply shall comply with any national regulations. The user shall be made aware of that, if the NucleoCounter and the external power supply is used in a manner not specified by the manufacturer, the protection provided by the NucleoCounter and the external power supply may be impaired.
  • Page 65 0004 (W x D x H – 120mm x 173mm x 102mm). The Thermo Printer PCNEOS-S2BN ChemoMetec part no. 939-0006 has identical dimensions. The NucleoCounter is equipped with a printer output port. It is located just below the USB connector on the rear side as apparent from Figure 34 and Figure 35...

  • Page 66: Nucleocounter Test Kit

    10.6 NucleoCounter Test Kit Figure 37 The NucleoCounter test kit. The NucleoCounter test kit consists of 3 levels of bead concentration and a blank. The test kit can bu used to test the performance of a NucleoCounter. Please refer to the package...
  • Page 67 10 Equipment and Accessories Figure 38 NucleoView The NucleoView PC software is used for handling of the obtained cell counts. The program is installed on a Windows PC (Win2000, Win98 2.edt., WinME, WinXP). A USB driver and the program are on the installation CD-ROM.

  • Page 68: Appendix 1: Result Mode 4

    11 Appendix 1: Result mode 4 11 Appendix 1: Result mode 4 The table below provides an overview of the results displayed when mode 4 (statistical table) is chosen compared to mode 3 (maximum precision). Lower limit Upper limit Lower limit Upper limit Mode 4 Mode 4...
  • Page 69 11 Appendix 1: Result mode 4 1.826 x 1.875 4.51 x 10E5 4.70 x 10E5 4.6 x 10E5 10E6 10E6 1.85 x 10E6 1.876 x 1.925 4.71 x 10E5 4.90 x 10E5 4.8 x 10E5 10E6 10E6 1.90 x 10E6 1.926 x 1.975 4.91 x 10E5...

  • Page 70: Appendix 2: Extended Nucleocounter Nc-100 Tm Settings

    “Save parameters?” when you turn it off. Push setting permanently. The NucleoCounter NC-100 has 9 different levels of sensitivity, which can be adjusted to count the different types of cells. Available in firm ware 4.10 and forward.
  • Page 71 ® 12 Appendix 2: Extended NucleoCounter NC-100 Settings Type 1 is the least sensitive (default NC-100) and should be used for the counting of mammalian cells. We recommend that NucleoView is used to select the best Type for your cells, by ensuring that cell signals are separated...
  • Page 72 ® 12 Appendix 2: Extended NucleoCounter NC-100 Settings This page was intentionally left blank...

  • Page 73: Appendix 3: Weee Directive Information In More Eu Languages

    13 Appendix 3: WEEE directive information in more EU languages United Correct Disposal of T his Product Kingdom (Waste Electrical & Electro nic Eq uip ment) - Europe onl y This marking sho wn on the product or its literature, indicates that it should not be disposed together with other household w astes at the end of its working life.
  • Page 74 Norwa y Korrekt a vhendi ng a v dette produkt (Avfall elektrisk og elektronisk utstyr) - Kun Europa Denne merkingen so m vises på produktet elle r dens doku mentasjon, indikerer at den ikke skal kastes sa mmen med annet husholdningsavfall ved slutten av sin levetid.
  • Page 75 Finland T ämän tuotteen turvalli nen hä vittämi nen (elektroniikka ja sä hkölaitteet) - Vain Eurooppa Oheinen merkintä tuotteessa tai tuotteen oheismateriaalissa merkitsee, että t ätä tuotetta ei tule hävittää kotitalousjätteen mukana sen elinkaaren päätyttyä. Hallitsematto masta jätteen käsittelystä ympäristölle ja kanssaihmisten terveydelle aiheutuvien vahinkojen välttä miseksi tuote tulee käsitellä...
  • Page 76 ChemoMetec affiliated companies shall held liable for damages and customers shall indemnify ChemoMetec A/S and affiliated companies against liability flowing from use of  potentially inaccurate data generated by the NucleoCounter . It recommended that results obtained with ...

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