NucleoCounter User's Guide P/N 991-0009 Revision 1.
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NucleoCounter Mammalian Cell Counter P/N 991-0009 (English) Revision 1.2 November, 2013 ChemoMetec A/S Gydevang 43, DK-3450 Allerød, Denmark Telephone: (+45) 48 13 10 20, Fax: (+45) 48 13 10 21 Internet: www.chemometec.com, E-mail:info@chemometec.
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Caution! This equipment must be operated as described in this User’ s Guide. Please read the entire guide before attempting to use this unit. Please pay attention to that gloves or protective clothing are not worn on the illustrations/pictures shown in this User’s Guide. However, ChemoMetec A/S does recommend that the user wear suitable protective clothing etc.
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Declaration of Conformity NucleoCounter Name of product: 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 – EMC requirements (emission and immunity). Annex B and C from A1: 1998 is used.
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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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Introduction and intended use Introduction and intended use ® The NucleoCounter is a novel instrument designed specially to count mammalian cells. The instrument is a part of the NucleoCounter system, which also comprises the NucleoCassette™, Lysis buffer (Reagent A100) and Stabilizing buffer (Reagent B). The NucleoCounter system allows reliable, fast and objective cell counting to be carried out based on the method of fluorescence microscopy.
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Warnings and precautions Warnings and precautions Wherever the symbol appears on the NucleoCounter instrument, it indicates that the manual must be consulted for precautions and warnings. 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.
Warnings and precautions Table 2 Description of the NucleoCounter Printer Output connector Name Maximum Voltage level 2 2 Rx ±10 VDC 3 Tx ±10 VDC Metal Enclosure DGND 0 VDC Pin no 13 2 3 1 Metal Enclosure 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.
Warnings and precautions 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. Electromagnetic interference NOTE: This equipment has been tested and found to comply with the limits for a Class B digital device, pursuant to Part 15 of the FCC Rules.
Warnings and precautions Never pipette by mouth. Avoid cross contamination of the samples when preparing of the samples. This can compromise the quality of the results.
1 Inspection and Unpacking of Equipment Table of contents DECLARATION OF CONFORMITY ................................................................................ VI WEEE DIRECTIVE INFORMATION – EUROPE ONLY ...................................................... VIII INTRODUCTION AND INTENDED USE .......................................................................... X WARNINGS AND PRECAUTIONS ...............................................................................
1 Inspection and Unpacking of Equipment 5.4.3 5.4.4 5.4.5 5.4.6 5.5 6 Setting Date (F200) and Time (F201) ......................................................... 18 Reset Counter (F30) ................................................................................... 18 LCD contrast (F220) ................................................................................... 19 Instrument Info (F100) ..............................................................................
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. Unpack the order, saving the packing materials for possible later use. Also be sure to save the User’s Guide, for instruction and reference.
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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.
2 Introducing the NucleoCounter 2.1.1 Fluorescence microscope The compact fluorescence microscope integrated in the NucleoCounter comprises LED´s (light emitting diodes) as excitation light source, excitation and emission filters, optics (lenses) and a CCD (charged coupled device) camera. Furthermore, the NucleoCounter contains advanced software for image processing. The fluorescence microscope, with a cassette inserted, is shown in Figure 3. Figure 3 .
2 Introducing the NucleoCounter A fluorescent image of the content of the measurement chamber is thus achieved. Example of such an image is shown in Figure 4. Each spot in the image represents a cell containing DNA stained with PI. Figure 4 . Fluorescent image (from NucleoView PC software) of cell 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.
2 Introducing the NucleoCounter Figure 5 The NucleoCassette. The fluorescent dye, propidium iodide, is immobilized in the first part of the flow system. 2.2.1 Loading the cassette The NucleoCassette is loaded by gently pressing the white piston, which creates a partial vacuum in the flow system. The tip of the NucleoCassette must be immersed into a lyzate mixture when pressing the piston, resulting in lyzate being loaded into the flow system. Approximately 60µl is loaded into the flow system. 2.2.
2 Introducing the NucleoCounter by multiplying the imaged area with the depth of the cassette. The imaged area is only dependent on the optics of the instrument. Therefore, the area is constant and specific for each instrument. 2.2.
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3 Control Buttons, Keypad and Display 3 Control Buttons, Keypad and Display 3.1 3.1.1 Interactive Controls Control Buttons There are four Control Buttons on the front of the instrument (see Figure 6 ): Table 3..
3 Control Buttons, Keypad and Display 3.2 3.2.1 Interface Display Status indicator A small Status Indicator light above the “On/Off” button (see Figure 6) illuminates when the unit is turned on. It is green when the instrument is ready to measure and it turns red while the instrument is measuring or displaying an error message. The Status Indicator turns green after successful measurement when the results are displayed. 3.2.2 Display screen The display screen communicates information to the user.
4 Installation and Start-up 4 Installation and Start-up 4.1 Power on, power off The NucleoCounter is powered by a 12VDC external power supply. To connect the power supply to the NucleoCounter, connect it to the DC socket (see Figure 7), and plug the mains power cord of the power supply into a wall outlet. 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.
4 Installation and Start-up After a successful start-up the display changes (Figure 9) and the status indicator on the keyboard panel turns green. NucleoCounter Ready Figure 9 . The NucleoCounter is ready for cell counting If the instrument has performed as described above, it is ready for use. If the instrument displays an error message at start-up please refer to chapter 7. 4.1.
5 Operation of the NucleoCounter 5 Operation of the NucleoCounter 5.1 Ready mode After the NucleoCounter has started successfully the display should read: NucleoCounter Nucl eoCounter Ready Figure 11. 11 . The NucleoCounter is ready for cell counting. This display is also shown if the “Esc” button is activated in the result mode or any other situation, except during measuring or shutting down. 5.
5 Operation of the NucleoCounter 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. Figure 12. 12 . Illustration of how to grip and insert the cassette into the insertion slit 5.3 Measuring - The Run button When a cassette has been loaded and inserted into the instrument, the measurement is activated, by pressing the Run button. 5.3.
5 Operation of the NucleoCounter 5.3.3 External computer If an external computer with the NucleoView software installed on it is connected, the results and the recorded fluorescent image can be viewed using the NucleoView software. After the measurement is completed the cassette can be safely removed (see section 5.2). Sometimes, however, the complete process described above may be interrupted. Please familiarize yourself with possible error handling of the NucleoCounter presented in chapter 8. 5.
5 Operation of the NucleoCounter • 5.4.2 Press <↵>. Saving parameters The representation of the results is changed with effect from the next analysis. When the NucleoCounter is turned off, after changing the status, the display will read “Save parameters?”, "ENTER: Save" and "ESC : Cancel" (Figure 14). Save parameters? ↵ ESC : Save : Cancel Figure 14 The shut-down display when any parameters has been changed.
5 Operation of the NucleoCounter 5.4.5 LCD contrast (F220) When typing “FF + 2 + 2 + 0 + ↵ ” it is possible to adjust the contrast of the LCD display. Use the “1 1 ” or “2 2 ” keys in order to increase or decrease the contrast of the LCD-display. The default value is 4. Values in the range from 0-9 can be chosen. The display will be updated with the current value, which is shown in the upper right hand corner of the display. Contrast? 1: Up 2: Down ↵ store 4 Figure 15. 15 .
5 Operation of the NucleoCounter in line 3. The serial number of the instrument is shown in line 4 of the display. The display text is almost identical to the start-up display text (Figure 17). 5.5 Zero Count Check (F50) The instrument can perform a so-called Zero Count Check (only firmware v 3.04 or higher). This check is carried out without cassette in the insertion slit of the instrument and with the lid closed. The check determines the number of CCD-pixels that show elevated signal levels.
5 Operation of the NucleoCounter Press “↵ ↵ ” to continue. Then a “Wait” message appears in the display: wait... Figure 21 Zero Count Check, step 3A After a few seconds the instrument has performed the Zero Count Check. If no contamination of the insertion slit can be detected an “OK” message appears in the display (see the figure below). Press any key to return to “Ready mode”.
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6 Total and viability count 6 Total and viability count This chapter describes the basic procedures for the determination of the total concentration of cells, the concentration of non-viable cells and calculation of viability in a cell sample. Furthermore various alternatives for diluting the samples are presented. The fundamentals on which the NucleoCounter is based are discussed thoroughly in chapter 2 above.
6 Total and viability count available to load the NucleoCassette. It is important that the sample must be representative of the cell suspension. Throughout the examples in this User’s Guide a sample size of 200 µl is used. Add 200 µl of the Lysis buffer to the cell sample and mix thoroughly by turning the vial upside down 5-10 times. Alternatively you can use the pipette. Add 200 µl of the Stabilizing buffer and mix thoroughly. This mixture is called the Stabilized lyzate .
6 Total and viability count After approximately 30 seconds the NucleoCounter shows the result in the display and on the computer if one is connected. Examine the result of the analysis. The measurement range of the NucleoCounter is 5•10 3 - 2•10 6 cells/ml. If the cell concentration of the stabilized lyzate is outside the measurement range then the cell suspension has to be concentrated (e.g. by centrifugation) or diluted (see section 6.4 below). The measurement is then repeated.
6 Total and viability count 6.3 Calculate viability When both the concentration of non-viable cells and the total concentration of cells are known, it is possible to calculate the viability (%viability) of the cells. The calculation can either be done manually or by use of the NucleoView software (please refer to the NucleoView TM User´s Guide). For the calculation of %viability the multiplication factors must be taken into account according to the equation shown below.
6 Total and viability count buffer added to the samples are altered. Note that it is important to add equal volumes of Lysis buffer and Stabilizing buffer to the samples. For examples of dilution using the Lysis buffer and Stabilizing buffer, see the table below.
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7 Maintenance of NucleoCounter 7 Maintenance of NucleoCounter Cleaning on a regular basis of the NucleoCounter is recommended in order to protect its surface and assure the quality of the collected images. 7.1 Cleaning Depending on the environment in which the NucleoCounter is operated it is suggested that regular cleaning of the cabinet is carried out. When cleaning the cabinet it is recommended to use a soft moist cloth and gently wipe the surface.
7 Maintenance of NucleoCounter Figure 25. 25 . 7.1.3 An image showing a contamination of the optical system, visible as a white cloudy phenomena in the upper right hand corner. If the contamination is stationary it should be removed using compressed air. Removal of dust particles The presence of a contaminant (e.g. dust particles) will normally not influence the counting of cells.
7 Maintenance of NucleoCounter 7.1.4 Spill of liquid If liquid has been spilled on the instrument it can contaminate elements of the optical system. This contamination can show up in the image in several ways and have many causes but a common feature is that it cannot be removed by the use of compressed air. Even though such phenomena are clearly visible on the image it only rarely affects the results of the NucleoCounter.
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8 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. 8.1 No valid cassette When trying to analyze a cassette, which is not valid, the NucleoCounter displays the error message shown in Figure 27. No valid cassette Press any key Figure 27. 27 .
8 Troubleshooting - Error messages 8.3 Actuator error messages The actuator moves the piston down into the barrel of the cassette. Two different actuator error messages can be displayed. These error messages are shown in Figure 29. The same action is to be taken if either of the error messages appears. Actuator error Refer to manual Press any key Figure 29. 29 . The NucleoCounter can display this message regarding the actuator First, examine if the cassette has been inserted properly.
8 Troubleshooting - Error messages Sensor error Refer to manual Press any key Figure 31. 31 . This error message is displayed if one of the sensors, which read the black dots on the cassettes, causes error. If the error message is displayed analyses can still be made but the accuracy of the results cannot be guaranteed since the volume of the measurement chamber may not be read correctly. 8.
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9 Technical specifications 9 Technical specifications 9.1 The NucleoCounter Specificity The NucleoCounter counts mammalian cell nuclei stained with the DNA specific fluorescent dye, propidium iodide. Sample consumption 100 µl is recommended for NucleoCassette loading. Approximately 60 µl is loaded into the cassette. Analysis volume Approximately 2 µl of the sample loaded into the NucleoCassette is transported to the chamber and is analyzed.
9 Technical specifications INSTALLATION CATEGORY II (Refer to UL 61010A-1) OVERVOLTAGE CATEGORY II (Refer to UL 61010A-1) POLLUTION DEGREE II (Refer to UL 61010A-1) 9.2 The NucleoCassette™ Reagent Each NucleoCassette contains approximately 2,8 µg propidium iodide. Storage Store the NucleoCassettes in a sealed foil bag at max. 30°C. Stability See documentation delivered with the NucleoCassettes. 9.3 EMC/EMI standards The NucleoCounter complies with EMC/EMI standards as follows.
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. Item Part no at ChemoMetec A/S Description NucleoCounter 900-0004 The NucleoCounter instrument NucleoCassette, 100 pcs. 941-0002 5 A disposable device used for performing the cell-count.
10 Equipment and Accessories Item Part no at ChemoMetec A/S Description current. Mains Power cord See section below. A cord for mains supply of the external power supply. Dry, compressed air See section below Used for removal of dust from the optical system of the NucleoCounter Dispenser 0.05-0.
10 Equipment and Accessories 10.3 Mains Power cord Detachable power supply cord set for the Class I specified power supplies: For US 125 Vac UL listed, type SVT, rated min. 60C, 18 AWG, 3 conductors. Provided with molded-on grounding-type (NEMA 5-15P) attachment Plug, rated min. 125 Vac, min. 2.5A. Opposite end terminates in molded on, IEC320 style connector, rated min. 125 Vac, min. 2.5 A. For Europe 250Vac Cord type min. H05RR-F or min. H03VV-F or min. H03VVH2-F, rated min. 60C, 3x0.75 mm2.
10 Equipment and Accessories The item can be obtained from Kenro Ltd, Greenbridge Road, Swindon SN3 3LH, UK. Tel: +44 (0) 1793 615836, E-mail: sales@kenro.co.uk. Alternatively the item, or a similar item, might be available through local dealers. 10.5 External Printer (optional) An external impact or thermo printer may optionally be connected to the NucleoCounter for the printing of test results. Figure 33 Impact printer type DP24-S1N ChemoMetec part no. 939-0004 (W x D x H – 120mm x 173mm x 102mm).
10 Equipment and Accessories Table 4 Description of the NucleoCounter Printer Output connector Pin no Name Maximum Voltage level 6 17 2 Rx ±10 VDC 3 Tx ±10 VDC Metal Enclosure DGND 0 VDC 2 3 1 Metal Enclosure Figure 36 Printer Output connector seen from the cable entry side. 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.
10 Equipment and Accessories package insert of the NucleoCounter test kit for operation and information on how to apply the kit in a test of a NucleoCounter. 10.7 NucleoView 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.
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 (mode 3) Up per limit (mode 3) Mode 4 Lower limit (mode 3) Upper limit (mode 3) Mode 4 N/A N/A < 5 x 10E3 5.63 x 10E5 5.87 x 10E5 5.75 x 10E5 N/A 7 x 10E3 5.0 x 10E3 5.88 x 10E5 6.12 x 10E5 6.00 x 10E5 8 x 10E3 1.3 x 10E4 1.0 x 10E4 6.13 x 10E5 6.37 x 10E5 6.25 x 10E5 1.
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12 Appendix 2: Extended NucleoCounter ® NC-100 TM Settings 12 Appendix 2: Extended NucleoCounter ® NC-100 T M Settings Enabling of extended instrument sensitivity (F566 (F5661) 1) 8 The extended instrument sensitivity is activated by the user function F5661 (“F5661 + ↵”) on the keypad. Once you have activated it, it cannot be deactivated! Instrument Sensitivity (F11) The appropriate setting is selected by the user function F11 (“F11 + ↵”) on the keypad.
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13 Appendix 3: WEEE directive information in more EU languages United Kingdom Correct Disposal of T his Product (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 wastes at the end of its working life.
Norwa y Korrekt a vhendi ng a v dette produkt (Avfall elektrisk og elektronisk utstyr) - Kun Europa Denne merkingen so m vises på produk tet eller dens doku mentas jon, indikerer at den ikke skal kastes sa mmen med annet husholdningsavfall ved slutten av sin levetid. For å hindre mulig skade på miljøet eller menneske lig helse fra ukontrollert avfallsavhending, vennligst atskill dette fra andre typer avfall og resirkuler det ansvarlig for å fremme b ærekraftig gje nbruk av materielle ressurser.
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 mas ta jätteen käsittelystä y mpäristölle ja kanssaihmisten terveydelle aiheutuvien vahinkojen välttä miseksi tuote tulee käsitellä muista jätteistä erillään. Jäte on hyvä kierrättää raaka-aineiksi kestävän ympäristökehityksen ta kia.
The information contained herein is to the best of our knowledge accurate and complete. However cell species and cell environments may vary in property. Therefore systematic and/or NucleoCounter random deviation between estimates obtained by the and other cell counting methods may occur. As such, nothing contained or stated herein including results obtained from use of the NucleoCounter or NucleoCassette™ shall be construed to imply any warranty or guarantee.
