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Using the keypad, enter the appropriate volumes and concentrations needed for your experiment.
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The calculation will automatically update below the text fields. When completed, press

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Chapter 7

Calculators

Cell Splitting Calculator

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Countess

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3 FL Automated Cell Counter User Guide

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Summary of Contents for AMQAF2000

Page 1: ...For Research Use Only Not for use in diagnostic procedures Countess 3 FL Automated Cell Counter USER GUIDE Catalog Number AMQAF2000 Publication Number MAN0019567 Revision B 0 ...

Page 2: ...IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT Revision history Pub No MAN0019567 Revision Date Description B 0 28 January 2021 Add component to Contents Table Update Technical specification A 0 09 November 2020 New user guide Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the te...

Page 3: ...ith a new Connect account 11 Sign in to the instrument with an existing Connect account 13 CHAPTER 3 Templates 14 Templates screen 14 Template functions 15 Count parameters 15 Load a template 16 Create a template 16 Edit a template 16 Import a template 17 Export a template 18 Delete a template 19 CHAPTER 4 Slide preparation and operation 20 Recommendations 20 Load Countess Cell Counting Chamber Sl...

Page 4: ... Edit and save a new template 29 CHAPTER 6 Fluorescence assays 30 Count cell fluorescence 30 Overview 30 Count procedure 30 Next steps 33 View results 33 Results screen for cell fluorescence assays 33 Identify objects counted 34 Identify cells counted in fluorescence assays 34 Histogram count results 34 View histogram for cell fluorescence assays 34 Gate count results 36 Gating screen 36 Gate coun...

Page 5: ...ht cubes 51 Install or change an EVOS light cube 51 CHAPTER 11 Maintenance 53 Instrument care 53 General guidelines for care 53 Power supply 53 Clean the cell counter 54 Introduction 54 Clean the touchscreen 54 Clean the instrument case 54 Decontaminate the instrument 55 APPENDIX A Troubleshooting 56 APPENDIX B Technical specifications 59 Physical characteristics 59 Technical specifications 59 Opt...

Page 6: ...s 63 APPENDIX E CSV file format definition 65 Overview 65 APPENDIX F Safety 69 Chemical safety 70 Biological hazard safety 71 APPENDIX G Documentation and support 72 Customer and technical support 72 Limited product warranty 72 Contents 6 Countess 3 FL Automated Cell Counter User Guide ...

Page 7: ...Automated Cell Counter 1 each Power cord with 4 adapters for U S Canada Taiwan Japan Europe or UK 1 each Countess Cell Counting Chamber Slides 50 slides box 1 box Countess 3 FL Disposable Slide Holder 1 each Countess 3 FL Reusable Slide Holder 1 each Countess 3 FL Light Cube Removal Tool 1 each Countess 3 USB Drive 1 each Countess 3 FL Automated Cell Counter Quick Reference Card 1 each Wi Fi Dongl...

Page 8: ...tion to the brightfield channel the Countess 3 FL Automated Cell Counter can accommodate two interchangeable EVOS fluorescent light cubes page 64 enabling it to be used for multiple fluorescence research applications When equipped with EVOS light cubes the Countess 3 FL Automated Cell Counter can be used to perform fluorescence assays for cells in suspension including simultaneous counts of cells ...

Page 9: ...angeable slide specific carriers For more information see Chapter 4 Slide preparation and operation 4 Back panel Allows access to the optional EVOS light cubes and provides storage for the light cube tool and the reusable slide carrier The back panel is secured to the instrument by two captive turn fasteners 5 Power switch ON OFF rocker switch is the main power switch It is not necessary to use th...

Page 10: ...trument from the wall outlet Install the instrument 1 Unpack the instrument and place the instrument on a flat level dry surface 2 Remove the thin plastic protector film from the touch screen display 3 Plug one end of the power cord appropriate for your region into the instrument 4 Plug the power cord into the electrical outlet Be sure to use only the power cord supplied with your instrument Power...

Page 11: ...eate customized count preferences i e gate counts based on cell size brightness circularity and or fluorescence intensity See Load a template on page 16 4 To change instrument settings press Settings Settings allows you to update the Countess 3 FL instrument software customize instrument settings view instrument details export error logs and change up to two EVOS light cubes See Settings screen on...

Page 12: ...ng code to enter online in your Thermo Fisher Connect account 4 A completion screen will show Press Done You then return to the Home screen and are logged into your account Once set up you will then only need to sign in to your instrument account using your Username and PIN Chapter 2 Installation Sign in to the instrument with a new Connect account 2 12 Countess 3 FL Automated Cell Counter User Gu...

Page 13: ...count 1 Press Sign in from the Home screen 2 Select your Username from the drop down box Enter your PIN 3 Press Sign in Chapter 2 Installation Sign in to the instrument with an existing Connect account 2 Countess 3 FL Automated Cell Counter User Guide 13 ...

Page 14: ...ount parameters are defined in the Edit template screen See Create a template on page 16 or Edit a template on page 16 The Default template contains default count settings and cannot be edited The count parameters specified in the selected template are applied to all new cell counts Templates can be sorted by pressing on the Name Channels or Last Modified headers If you have already performed a co...

Page 15: ...ng the bottom gate up removes smaller objects moving it down includes smaller objects 2 Brightness Gating Moving the top gate down removes brighter objects moving it up includes brighter objects Moving the bottom gate up removes dimmer objects moving it down includes dimmer objects 3 Cicularity Gating Moving the top gate down removes more circular objects moving it up includes more circular object...

Page 16: ...xt box to open the keypad 3 Type in the desired template name To enter symbols press the symbol key To return to the alpha numeric keypad press ABC 4 Press Enter to save the name and return to the New Template screen Press to return to the New Template screen without saving the name 5 Press Save to save the new template Press Cancel or Back to return to the Templates screen without saving Edit a t...

Page 17: ...late and press Load to use that template to read the sample Import a template 1 Press Templates 2 Press Actions 3 Press Import 4 Press the import location Cloud or USB a For Cloud press Sign in to log in to your Thermo Fisher Connect account If the Cloud is not active and disconnected press Network settings to connect to a network then press Back to try and sign in again Chapter 3 Templates Import...

Page 18: ...s Actions 3 Press Export 4 Select one or more templates from the list 5 Press Next 6 Press the export location Cloud or USB a For Cloud press Sign in to log in to your Thermo Fisher Connect account If the Cloud is not active and disconnected press Network settings to connect to a network then press Back to try and sign in again Chapter 3 Templates Export a template 3 18 Countess 3 FL Automated Cel...

Page 19: ... Delete a template 1 Press Templates 2 Press Actions 3 Press Delete 4 Select one or more templates from the list 5 Press Delete The system will prompt you to confirm the deletion then press Delete again 6 Once complete press Done Chapter 3 Templates Delete a template 3 Countess 3 FL Automated Cell Counter User Guide 19 ...

Page 20: ...e that the counting area is covered with the cell suspension and count the cells immediately after staining per the assay protocol Do not press the optical surfaces of the chamber slides Hold the slides by the edges Take care to avoid forming bubbles in the sample Load Countess Cell Counting Chamber Slide 1 Prepare the sample by adding 10 µL of your cell suspension to 10 µL of 0 4 trypan blue stai...

Page 21: ... Countess Cell Counting Chamber Slides appropriately dispose of them as biohazardous waste Do not reuse the disposable chamber slides 6 Optional To remove the slide adapter gently squeeze the tabs and pull the adapter completely out of the instrument Load the Countess Reusable Slide 1 Before loading your sample into the Countess Reusable Slide place a cover slip on the counting chamber making sure...

Page 22: ...ss Cell Counting Chamber Slide or the Countess 3 FL Reusable Slide has a 10 µL sample capacity Do not overfill the slide chambers 4 To use the Countess Reusable Slide unlatch the back panel of the Countess 3 FL Automated Cell Counter with the two captive turn fasteners that secure the back panel on the rear of the instrument Chapter 4 Slide preparation and operation Load the Countess Reusable Slid...

Page 23: ...slide out Grasp the slide and pull it out the rest of the way 10 Optional To count the second sample present on the reusable slide simply remove the slide from the holder rotate and reinsert the slide into the holder so that the second sample is aligned with the sample viewing hole 11 After using the Countess Reusable Slide rinse the glass slide and cover slip with water and then clean with 70 eth...

Page 24: ...er settings as described in Load a template on page 16 3 Insert the sample slide into the slide port making sure that the sample side is inserted completely into the instrument You will hear a soft click if the slide is pushed in correctly 4 When the slide is inserted the instrument automatically illuminates the sample sets the intensity of brightfield illumination and auto focuses on the cells No...

Page 25: ...ty assays on page 27 Note The Live and Dead buttons are enabled by default after the image has been counted To see the distribution of live and dead cells in a graphical format press Histogram See Histogram count results on page 28 To gate the results by object size brightness or circularity press Gating to go to the Gating screen See Gate count results on page 28 Note You can save the changes you...

Page 26: ...rypan blue and takes this dilution into account when calculating the total cell concentration The cell concentration displayed in the Results screen is the original cell concentration before dilution into trypan blue Identify objects counted Results screen The Results screen allows you to identify the objects i e cells counted in each channel and included in the count results for further review Af...

Page 27: ...ailed image below shows both Live green and Dead red cells are selected 3 To unmark the cells press the appropriate colored button again Individual cells can be selected to view their specific size brightness and circularity gating settings Zooming in on the image will allow for easier selection of individual objects Click on the desired cell and a pop up displays the parameters Press to close the...

Page 28: ...n size only in the selected population 3 Optional Press Gating Using the Size Brightness and Circularity sliders adjust the count parameters As you adjust the count parameters the count results and the graph will be automatically updated 4 To close the histogram press Histogram or Gate count results Gating screen The Gating screen for cell count and cell viability assays in the brightfield channel...

Page 29: ...unt you may save your settings to a new template 1 Press Templates 2 To create a new template with the edited parameters press Create The new template screen opens and displays the edited count parameters from the Gating screen 3 To update an existing template with the edited parameters select a template from the list then press Actions4Edit Note You cannot edit the Default template The Edit Templ...

Page 30: ...n the Templates screen and load the desired gating parameter as described in Load a template on page 16 4 Insert the sample slide into the slide port Exterior instrument parts on page 9 making sure that the sample side is inserted completely into the instrument You will hear a soft click if the slide is pushed in correctly 5 When the slide is inserted the instrument automatically illuminates the s...

Page 31: ...ss Adjust to go to the Adjust screen 8 Press the light source button for the channel you wish to set exposure and adjust the exposure using the light source slider Repeat the procedure for the remaining channels if desired 9 Press Apply to keep the current setting and return to the Setup screen OR press Cancel or Back to return to the Setup screen without saving changes Chapter 6 Fluorescence assa...

Page 32: ...s the images and displays the results total concentration percentage and concentration of cells counted in each fluorescence channel For more information see View results on page 33 Autosaving will occur when the count is completed if you have Autosave enabled from the Save Options screen Chapter 6 Fluorescence assays Count cell fluorescence 6 32 Countess 3 FL Automated Cell Counter User Guide ...

Page 33: ...unt results press Calculators to open the Calculator application See Chapter 7 Calculators To permanently save the results press Save See Save screen on page 41 To perform a new count push the slide to eject then insert a new sample slide View results Results screen for cell fluorescence assays The Results screen for cell fluorescence assays displays a composite image of the objects counted and th...

Page 34: ... and the cells counted in the DAPI and TX Red channels are marked with blue and red circles respectively 3 To unmark the cells counted in a specific channel press the corresponding boundaries button again Histogram count results View histogram for cell fluorescence assays For fluorescence assays you have the option of viewing the distribution of the cells based on size or based on relative fluores...

Page 35: ...press the corresponding channel button on the histogram The histogram automatically updates and displays the distribution of cells based on relative fluorescence intensity only in the selected i e checked channel 4 To add the cells counted in a specific channel to the histogram press the corresponding channel button 5 To close the histogram press Histogram or Close Chapter 6 Fluorescence assays Hi...

Page 36: ...o view the distribution of cells based on size or fluorescence intensity See View results on page 33 3 Press BF brightfield to adjust the parameters for size brightness and circularity using the gating sliders 4 Select the desired fluorescence channel e g DAPI TX Red etc to adjust the threshold for fluorescence intensity using the fluorescence intensity slider Note The fluorescence channels availa...

Page 37: ... screen opens To import the current settings from the Gating screen press Import settings 4 To change the name of the selected template press the Template name text box and enter the desired name using the alphanumeric keypad 5 Optional If desired make additional changes to the template and the count parameters 6 Press Save to save the new template settings and return to the Templates screen Press...

Page 38: ...ield count this calculator also has an option to account for trypan blue use 1 On the Results screen press Calculators then select Pre Dilution Calculator 2 Click in one of the text fields Note For BF brightfield counts using trypan blue you can select the checkbox at the bottom of the screen to correct for a 1 1 trypan blue dilution 7 38 Countess 3 FL Automated Cell Counter User Guide ...

Page 39: ...ll appear at the top of the Results screen Cell Splitting Calculator The Cell Splitting Calculator is used to determine the amounts of sample and buffer needed to reach a desired concentration 1 On the Results screen press Calculators then select Cell Splitting Calculator Chapter 7 Calculators Cell Splitting Calculator 7 Countess 3 FL Automated Cell Counter User Guide 39 ...

Page 40: ... and concentrations needed for your experiment Press Enter after filling in the text boxes 4 The calculation will automatically update below the text fields When completed press Done Chapter 7 Calculators Cell Splitting Calculator 7 40 Countess 3 FL Automated Cell Counter User Guide ...

Page 41: ...format Note A dropdown list will provide options for how many counts to save You can choose from Current Capture Session Today Yesterday Past 7 days Past 30 days or All FCS Saves the data from the experiment as a Flow Cytometry Data File Standard The FCS format allows for processing or re displaying results with FlowJo or flowCore commonly used tools for flow cytometry Note If you wish to save you...

Page 42: ... the Save screen without saving the name press Close 5 Select the desired file type to save your experiment Result Images CSV Report Cell Data You can select an individual mode e g Result only or any combination of modes e g Result Images Data and or Report In the example below Report and CSV are selected 6 By default Result and Images are saved as TIFF files and CSV uses the current capture data ...

Page 43: ...ays or All After you make your selection the instrument returns to the Save screen To return to the Save screen without changing the file format press Back 8 Press Save to save your experiment to the selected mode s in the USB drive or Connect account 9 Press Done and then transfer the USB drive to the desired location Chapter 8 Save results Save procedure 8 Countess 3 FL Automated Cell Counter Us...

Page 44: ...ration of the sample and the percentage and number for the total Live and Dead channels Below the results table the report contains the number of cells vs cell size graph Under the graph the report contains the brightfield count image with the live and dead cells identified by the green and red circles respectively At the top the report displays the template information used to gate these images C...

Page 45: ...aphs the report contains the count images with the brightfield image on the left and the fluorescence images on the right In the brightfield image the cells counted in the brightfield channel are identified by the white total count circles In the fluorescence image overlaid if there are two channels the cells counted are identified by the circles with the same color as the fluorescence channel in ...

Page 46: ... an EVOS light cube on page 51 Connect to a wireless network Network connecting options 1 From the Home screen press Settings 2 Press Instrument settings 3 Press Network settings A list of available networks will display 4 Select the desired network and press Connect 1 From the Home screen press Save options 2 Press Network settings 3 Select the desired network and press Connect Follow either set ...

Page 47: ...saved on the top level of the USB drive not within a folder or a subfolder The software update file must be uncorrupted during transfer Do not rename zip or compress the software update file Update the Countess 3 3 FL Cell Counter software 1 Go to www thermofisher com countessupdate and download the latest Countess 3 3 FL cell counter software version to your desktop Note The software update file ...

Page 48: ...he Home screen will appear to resume normal operation Set the date and time 1 Press Settings on the Home page 2 Press Instrument Settings 3 Press Date and time 4 Select the date format Month Day Year Day Month Year or Year Month Day 5 Press any Date text box Month Day or Year to open the Edit Date keypad 6 Using the keypad enter the date into the Month Day and Year text boxes Press Enter Chapter 9...

Page 49: ...with AM or PM or 24 Hours 10 Press Apply to set the Time and Date and return to the Instrument Settings screen Press Cancel or Back to return to the Instrument Settings screen without saving your changes Change your cloud region 1 Press Settings on the Home page 2 Press Instrument Settings Chapter 9 Settings overview Change your cloud region 9 Countess 3 FL Automated Cell Counter User Guide 49 ...

Page 50: ...ct the desired cloud region from the available choices Press Change 5 Once the change is complete press Done Note Changing the cloud region will require relinking of all instrument accounts Chapter 9 Settings overview Change your cloud region 9 50 Countess 3 FL Automated Cell Counter User Guide ...

Page 51: ...ettings on the Home page 2 Press Instrument Settings 3 Press Change light cube The instrument positions the light cube tray to enable light cube installation 4 When prompted power off the Countess 3 FL Automated Cell Counter using the power switch on the back of the instrument Exterior instrument parts on page 9 5 Unplug the power cord from the Countess 3 FL Automated Cell Counter 6 Unlatch the ba...

Page 52: ...be removal tool into the central hole in the cube white arrows in Figure B and gently pull the light cube out of the device Note Always store the cube removal tool in the back panel for easy access 11 Install the back panel and secure it in its place with both turn fasteners 12 Plug the power cord back into the Countess 3 FL Automated Cell Counter 13 Turn on the Countess 3 FL Automated Cell Counte...

Page 53: ...incompatible power adapter is not covered by warranty CAUTION Never disassemble or service the instrument yourself Do not remove any covers or parts that require the use of a tool to obtain access to moving parts Operators must be trained before being allowed to perform the hazardous operation Unauthorized repairs may damage the instrument or alter its functionality which may void your warranty Co...

Page 54: ...ounter using a soft lint free cloth moistened with an LCD cleaning solution Do not apply excessive force during cleaning Wipe the touchscreen dry immediately after cleaning Ensure that the cleaning solution does not enter the power button the power inlet the slide port or the USB ports Never pour or spray any liquids directly on the instrument to avoid electrical shock when the instrument is plugg...

Page 55: ... solution because it may leave a residue of bleach crystals on the instrument Ensure that water or other cleaning solutions do not enter the power button the power inlet the slide port or the USB ports Never pour or spray any liquids directly on the instrument to avoid electrical shock when the instrument is plugged in Chapter 11 Maintenance Clean the cell counter 11 Countess 3 FL Automated Cell C...

Page 56: ... the count Brightfield or fluorescence settings were not optimal For cell count and cell viability assays performed in brightfield adjust the size brightness and circularity gates for both live and dead cells to include all of the cells in the count For fluorescence assays adjust the size brightness circularity and fluorescence intensity gates in all available channels to include all of the cells ...

Page 57: ...eld of view each time a slide is inserted Depending on the concentration and uniformity of the cells this will cause some variability when performing replicate counts of the same slide although it should be less than 10 When counting fewer cells a small field of view change for only a small number of cells can have a larger affect Count cells at a higher concentration to reduce variability Make su...

Page 58: ...d USB drive Unable to update the Countess software USB drive did not function properly Make sure the USB drive is formatted to FAT32 If it is not reformat the USB drive to FAT32 before transferring the files onto the USB drive for software update Make sure the update file is at the top root of the USB drive not in a folder or subfolder File cannot be renamed in any way File cannot be zipped or com...

Page 59: ...t industrial Pollution degree 2 Operating temperature 10 40 50 104 F Operating humidity 80 non condensing Technical specifications Processing time 30 seconds Sample concentration range 1 104 1 107 cells mL Particle cell diameter range 4 60 µm Required sample volume 10 µL Firmware Countess Automated Cell Counting Platform Software USB drive 32 Gigabytes FAT32 exFAT or NTSF compatible Optics Optics ...

Page 60: ...is slide Material Poly methyl methacrylate PMMA Dimensions 25 mm W 75 mm D 1 7 mm H Chamber volume 10 µL Appendix B Technical specifications Analysis slide B 60 Countess 3 FL Automated Cell Counter User Guide ...

Page 61: ... LED collimating optics and filters In addition to the brightfield channel dedicated to cell count and cell viability assays using trypan blue the Countess 3 FL Automated Cell Counter can accommodate two fluorescent light cubes for multiple fluorescence research applications The following table lists some of the common fluorescent and specialty EVOS light cubes available from Thermo Fisher Scienti...

Page 62: ...lexa Fluor 647 Alexa Fluor 660 DRAQ5 Cy5 5 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 Alexa Fluor 700 Cy7 Cy7 IRDye 800CW Note The EVOS light cubes are available only for the Countess 3 FL Automated Cell Counter The Countess 3 Automated Cell Counter uses only brightfield illumination and does not support the EVOS light cubes Appendix C EVOS light cubes EVOS light cubes C 62 Countess 3 FL Automated Cell...

Page 63: ... 1 each A48208 Accessory products The following products can be used with the Countess 3 3 FL Automated Cell Counters and are available separately from Thermo Fisher Scientific For more information visit www thermofisher com or contact Technical Support Product Quantity Cat No Countess Cell Counting Chamber Slides 50 Slides 100 counts 1 box 1 C10228 Countess Cell Counting Chamber Slides 500 Slides...

Page 64: ...e stain 0 4 2 1 mL T10282 1 Each box of Countess Cell Counting Chamber Slides contains 50 slides and 2 1 mL vials of trypan blue 0 4 sufficient for 100 counts Appendix D Countess 3 FL Automated Cell Counter and accessories Accessory products D 64 Countess 3 FL Automated Cell Counter User Guide ...

Page 65: ...L Fluorescence Trypan Blue Brightfield F Trypan blue corrected Sample with trypan blue correction applied G Pre Dilution corrected Sample with pre dilution correction applied H Total concentration Concentration of the entire sample I Total cells counted Total number of cells counted in the sample J Live concentration Concetration of just the live portion of the sample K Live cells counted Live cel...

Page 66: ... of the total cells in brightfield that show fluorescence in the first and second cube positions combined AA Cube 1 2 cells counted Total number of cells counted in the first and second cube positions combined AB Cube 1 average Size µm Average size in microns of cells counted using the first top cube AC Cube 2 average Size µm Average size in microns of cells counted using the second bottom cube Ge...

Page 67: ...light intensity Second bottom light cube light intensity value from 0 100 AW Cube 2 LED intensity LED intensity of second bottom light cube Trypan Blue Brightfield AX BF size min Minimum size of brightfield cells in microns AY BF size max Maximum size of brightfield cells in microns AZ BF brightness min Brightfield adjustment slider value for minimum brightness BA BF brightness max Brightfield adj...

Page 68: ... Second bottom light cube adjustment slider value for maximum brightness General BH Template name Template used for count BI Software revision Current software version used for count Appendix E CSV file format definition Overview E 68 Countess 3 FL Automated Cell Counter User Guide ...

Page 69: ...tion provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection and so on To obtain SDSs see the D...

Page 70: ...s A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if needed the waste generated by the particular applicati...

Page 71: ...es Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in laboratory environment U S Department of Health and Human Services Biosafety in...

Page 72: ...protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale at www thermofisher com us en home global terms and co...

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Page 74: ...3 FL Automated Cell Counter_UG_MAN0019567 v3 GUID 2A84EC4A BB10 453C A0AD 5AA09BBA4C09 2021 01 28 19 40 13 en 20 14 40 199Z thermofisher com support thermofisher com askaquestion thermofisher com 28 January 2021 ...

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