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1. Assays amp Settings Select to return to Assay Selection Screen Sample ID Select to enter a Sample ID Dilution Factor Select to enter a Dilution Factor View Data File Opens data file to view or print View Size Histogram Opens separate window to display cell size histogram Save Copy of Data Select to saves the current counting results and images View Counted Image Shows cell image with green and red outlines to indicate cells counted Current View Red rectangle indicates what area of the cell image is currently displayed Pan Moves the area currently being shown within the current cell image Zoom Zooms in or out of the current cell image Return Select to return to Results Screen Current Image Select location on slide to view counted image O N WO OB ON a oi w N O SO 2013 Nexcelom Bioscience LLC 13 Assay Editor Main Screen 1 Current Assay Name of assay description and icon that appears in the Assay Selection screen 2 Imaging Mode Select to indicate which type of images are captured and analyzed 3 Channel 1 Indicates which Cell Type parameters are used based on the Imaging mode selected 4 Advance Settings Select to edit additional settings available for the imaging mode selected 14 2013 Nexcelom Bioscience LLC Assay Description Screen 2013 Nexcelom Bioscience LLC Imaging Mode Screen 1 Calculations and Reports Select to edit the report formats and the cal
2. button Take Background Image Used for initial setup of instrument and to auto adjust exposure time for bright field images a OO O F V Et Instrument Settings 4 Check the background image by clicking on the Cell Line Total Cell Conc assay and the Preview Image button The display should show an even white screen If any dark areas or variations are present contact Nexcelom Technical Support Cell line total cell conc No stain Cell line oc cultured primary cells without debris with viability greater than 98 20 2013 Nexcelom Bioscience LLC Auto Save Set up 1 Insert a USB drive or connect the Auto 1000 to a network drive via an Ethernet network cable 2 Click on the Settings button then the Saving Options button ne Saving Options Set sample ID naming convention Set location for data file Set location for images to be saved Tum on off auto printing of results l mn mt ms t Instrument Ut Settings 3 Click Auto Save to Data File and Save Raw Images to the On position Click on the File and Folder buttons to set the locations for saving In each case select the USB drive or network drive and name the folder Having saved images will help if any remote technical support is required and provides a permanent record of the raw data Click Done then click Done again Auto Save to Data File on E Save Raw Images crate rie trescr some
3. ay OF son Coated ge 4 Click Done then click Done again SS ae C Begin Next Sample Preparing Reference Beads 1 Though the Auto 1000 instrument does not require any routine testing or calibration reference beads are available 2 Cellometer Check Validation Bead Solution product number CCBM 011 2ML is recommended for use with the Auto 1000 3 Invert the reference bead solution a total of ten times 4 Vortex the bead solution for ten seconds 5 Set pipette to 20 ul 6 Pipette bead solution up and down ten times to break up any bead clumps 2013 Nexcelom Bioscience LLC 21 Counting Reference Beads 1 Peel plastic off of both sides of the Cellometer slide for PD100 slides the plastic has already been removed 2 Place cell counting chamber on a fresh Kimwipe 3 Load 20 ul of mixed Reference Bead Solution into the Cellometer Counting Chamber 4 Insert the loaded chamber into the Auto 1000 sample slot and gently push the slide to the stop 5 Select the Cell line total cell conc assay Cell line total cell conc Cell line of cultured primary cells without debris with viability greater than 98 6 Click on Preview Image for Current Assay 7 Adjust the focus if necessary using the coarse and fine adjustments on the left hand side of the screen until the best bead counting focus is achieved The beads should appear as dark circles with sharp edges e o Bae x o o o o Fi
4. screen View Counted Image View Details of Counting Results 24 2013 Nexcelom Bioscience LLC 9 Review the counted image Live cells should be circled in green Dead cells stained dark with trypan blue should be circled in red Q Q o o O 3 O Q O J Q 10 The Auto 1000 is now ready to analyze the next sample After inserting the imaging chamber loaded with the next sample click on the Next Sample button at the bottom right of the screen When prompted enter the Sample ID then click Count Begin Next Sample 2013 Nexcelom Bioscience LLC 25 Tutorials Overview The following tutorials are intended as a guide to performing various cell counting assays using the Auto 1000 General sample preparation hints are included for each tutorial as well as instrument and software operation instructions Each of the assays can also be performed using the sample images included in the software as a demonstration of the Auto 1000 Sample images for each cell counting assay can be found at C Program Files Nexcelom Assay_Images Staining Solutions Trypan Blue Stock solution 0 2 in PBS Use 1 1 with cell sample Dilution factor 2 Installed Assays and Descriptions Cell Line Total Cell Concentration No Stain Cell line or primary cells without debris with viability greater than 98 After taking brightfield images of a cell sample all cells are counted to determine total cell conce
5. L is optimal 2 Invert the tube containing cells ten times and pipette up and down ten times to generate a homogeneous cell sample and reduce cell clumps Do not shake or vortex the sample This will generate bubbles 3 For viability measurement stain cells by combining 50 ul of cell sample with 50 ul of a 0 2 trypan blue staining solution for a final concentration of 0 1 trypan blue Gently mix by pipetting up and down ten times 2013 Nexcelom Bioscience LLC 23 Cell Concentration and Trypan Blue Viability using the Auto 1000 1 Pipette up and down gently 10x to break any potential cell clumps in the stained cell sample 2 Load 20ul of sample into the Cellometer Counting Chamber CHT4 3 Insert counting chamber into instrument 4 Click on the Cell line viability trypan blue assay When prompted enter your desired Sample ID and a dilution factor of 2 Cell line viability trypan blue Trypan blue Cell line or cultured primary cells without debris 5 Click on Preview Image for Current Assay 6 Adjust the focus if necessary using the coarse and fine adjustments on the left hand side of the screen Cells in focus will have a bright center and crisp edge Fine Focus 7 Click the Count button at the bottom of the screen Count Current Image 8 Click on the View Details button at the bottom left of the screen then click on the View Counted Image button on the left hand side of the
6. culations used to generate results 16 2013 Nexcelom Bioscience LLC Channel Settings Screen 2013 Nexcelom Bioscience LLC Cell Type Library Screen 18 2013 Nexcelom Bioscience LLC Cell Type Parameters Screen 2013 Nexcelom Bioscience LLC Getting Started All Nexcelom products undergo a rigorous quality inspection prior to shipment and all reasonable precautions are taken in preparing them for shipment to assure safe delivery The instrument should be unpacked and inspected for mechanical damage upon receipt Mechanical inspection involves checking for signs of physical damage such as scratched dents etc If damage is apparent or any components are missing please immediately contact Nexcelom 1 978 327 5340 or support nexcelom com or your local dealer After unpacking the instrument plug the Cellometer Power Cable into the back of the instrument The Auto 1000 is pre configured with the Cellometer Auto 1000 software No additional setup or configuration Is required Starting the Auto 1000 After plugging in the system simply press the power button on the front of the unit Taking a Background Image 1 When using the Auto 1000 for the first time a background image should be taken This step does not need to be repeated unless the instrument is moved 2 Confirm that there is no slide in the Auto 1000 instrument 3 Click on the Settings button then take the Take Background Image
7. e only in conjunction with Nexcelom s family of products Limitation of Liability Hardware and Software Cellometer branded automatic cell counting instruments software and consumables are intended for research use only In no event shall Nexcelom be liable for any damages whatsoever including without limitation incidental direct indirect soecial or consequential damages damages for loss of business profits business interruption loss of business information arising out of the use or inability to use this Software Consumables or related Hardware 2013 Nexcelom Bioscience LLC 3 Nexcelom Bioscience LLC 360 Merrimack Street Building 9 Lawrence MA 01843 USA Phone 1 978 327 5340 Fax 1 978 327 5341 Email support nexcelom com www nexcelom com All content copyright 2013 Nexcelom Bioscience LLC Document 8001505 Rev A
8. help please call us at 1 978 327 5340 or email support nexcelom com for applications and other technical information sales nexcelom com for placing a purchase order or inquiries on a purchase order info nexcelom com for general inquiries 30 2013 Nexcelom Bioscience LLC Warranty Information Nexcelom warrants that Nexcelom instrumentation products shall for a period of 12 twelve months from the date of purchase be free of any defect in material and workmanship The sole obligation of this warranty shall be to either repair or replace at our expense the product at manufacturers option The original sales receipt must be supplied for warranty repair Products which have been subjected to abuse misuse vandalism accident alteration neglect unauthorized repair or improper installation will not be covered by warranty Any Product being returned is to be properly disinfected and packaged in original packing if possible Damage sustained in shipping due to improper packing will not be covered by warranty A valid Return Material Authorization Number RMA is required for all warranty repairs For RMA instructions please contact our customer service department at 978 327 5340 or email support nexcelom com License Agreement This agreement states the terms and conditions upon which Nexcelom Bioscience LLC Nexcelom offers to license to you the software together with all related documentations The Software is licensed to you for us
9. lity The Cellometer Auto 1000 system consists of 2 main components 1 Cellometer Auto 1000 instrument with analyzing software e Disposable counting chamber accommodates 2 individual samples and can be loaded through either port e Pipette 20uL of cell sample into one of the ports with any standard single channel pipette e Cellometer Auto 1000 comes with a starter set of 75 slides Slides can be ordered directly from Nexcelom or your authorized Nexcelom dealer 2013 Nexcelom Bioscience LLC Quick Operation Instructions 1 Select an Assay 2 Input Sample ID and Dilution Factor 3 Prepare Sample load disposable counting chamber and insert into instrument 4 Click Preview Coarse Fine Focus Focus 6 2013 Nexcelom Bioscience LLC 6 Select image to preview and adjust exposure if needed 7 Click Count fo begin counting process eS Se Count Current Image 8 Review Counting Results say Cell line total cell conc Sample ID Cell Line Dilution Factor 1 00 Mean Diameter 15 5 microns View Details of Counting Results 10 Select Next Sample or Assay and Settings when done Begin Next Sample 2013 Nexcelom Bioscience LLC User Interface Assay Selection Screen Cell line total cell conc Cell line or cultured primary cells without debris with viability greater than 98 Cell line viability trypan blue Trypan bue Cell line or c
10. n an ID that will be recorded with the data 4 Time Stamp Sample ID Selecting this will auto append the Sample ID with the date and time the count was performed 5 Include Instrument ID in File Selecting this will auto append the Instrument ID to the sample ID after the count is performed Auto Save 6 File this button brings up the File Dialog Window which allows the user to specify where the data txt file will be saved when Auto Save to Data File is selected 7 Auto Save to Data File Selecting this will auto save the data into the Data ixt file after a count is performed 28 2013 Nexcelom Bioscience LLC 8 Create New File for Each Sample Selecting this will auto create a new data ixt file for each sample and save the counting results to it after a count is performed 9 Folder this button brings up the File Dialog Window which allows the user to specify where the image folder will be saved when Save Raw Images or Save Counted Images is selected 10 Save Raw Images Selecting this will auto save the images to the folder specified using the Folder command 11 Save Counted Images Selecting this will auto save the counted images to the folder specified using the Folder command Auto Print 12 Auto Print Results Select to have the results sent to the printer Upon completion of count 13 Print with Default Printer Select to have the Windows default selected printer used when Auto Print Results is selected 14 Done Save Settings Click to
11. ne o Focus o 8 Click the Count button at the bottom of the screen Count Current Image 22 2013 Nexcelom Bioscience LLC 9 When counting is complete click on the View Details button at the bottom left of the screen View Details of Counting Results 10 Click on the View Counted Image button on the left hand side of the screen Enlarge the image by clicking the Zoom In button on the right hand side of the screen Confirm that all of the beads are circled in green e A View Counted Image Zoom In Out e L e Comparing Viability Methods Comparing Methods When evaluating different viability methods it is critically important to use one aliquot from the stock cell culture to perform all testing The cell sample should be evaluated for concentration on the Cellometer Auto 1000 prior to staining Trypan Blue Viability For cell lines and cultured primary cells bright field imaging and trypan blue viability may be used to determine the number concentration and percentage of live cells The trypan blue method is not recommended for samples containing debris platelets or red blood cells Fluorescence is required to accurately differentiate nucleated cells from platelets red blood cells and debris Preparing Cell Sample for Trypan Blue Viability Determination 1 A cell concentration of 1 0 x 10 to 1 0 x 10 cells mL can be analyzed on the Auto 1000 A concentration of 1 0 x 10 cells m
12. ntration Cell Line Viability Trypan Blue Trypan Blue Cell line or cultured primary cells without debris Trypan blue is routinely used to determine cell viability Trypan blue penetrates and stains dead cells and leaves live cells unstained After taking brightfield images of the stained sample live and dead cells are counted to determine total live and dead cell concentrations as well as compute percent viability 26 2013 Nexcelom Bioscience LLC Operation Reference Counting Options Speed Count Mel Use Celle Limit Stop after 1 Images Use Images Limit 1 Count All Select to have all 4 positions of slide used for cell images 2 Speed Count Select to have counting use Cell Limit or Image Limit a Use Cells Limit Check to stop counting after of User defined cells are counted and the next frame has finished counting b Use Images Limit Stop counting after finishing user defined images that are less than 4 images 3 Done Save Settings Saves the current settings and returns to previous screen 2013 Nexcelom Bioscience LLC 27 Saving Options Log User Name Save Options 1 Set Sample ID as Cell Type Selecting this will auto input the Sample ID to match the Cell Type parameter name being used for counting 2 Auto Increment Sample ID Selecting this will auto append the Sample ID with an incremental numerical value Example CHO sample_001 3 Log User Name Selecting this will require the user to enter i
13. peglab Cellometer Auto 1000 User Manual 2013 Nexcelom Bioscience LLC Cellometer Auto 1000 User Manual Introduction What is Cellometer Auto 1000 Quick Operation Instructions User Interface Overview Getting Started Starting the Auto 1000 Taking a Background Image Auto Save Set up Preparing Reference Beads Counting Reference Beads Comparing Viability Methods Preparing Cell Sample for Trypan Blue Viability Determination Cell Concentration and Trypan Blue Viability using the Auto 1000 Tutorials Overview Staining Solutions Installed Assays and Descriptions Operation Reference Counting Options Saving Options Technical Information Specifications Getting Support Warranty Information On oO Mm 20 20 20 21 21 22 23 25 24 26 26 26 26 27 27 28 30 30 30 3 Introduction What is Cellometer Auto 1000 Cellometer Auto 1000 is a compact automated cell counting system to count measure cell size and calculate cell concentration from a 20uL cell sample The basic principle of the Cellometer automatic cell counter is imaging cytometry Cells are loaded into the Disposable Counting Chamber and automatically soread into a thin layer by capillary action Cellometer Auto 1000 then captures images of cells in the counting chamber analyzes the number of cells and sizes of each cell and then converts this data into concentration size and viabi
14. save new settings and return to Assay and Settings main screen 2013 Nexcelom Bioscience LLC 29 Technical Information Specifications Size and Weight Height 9 0 inches 229 mm Width 11 1 inches 282 mm Depth 8 3 inches 211 mm Weight 9 0 Pounds 4 1 kg Environmental Requirements Typical biology lab environment Display 10 4 inch 1024 x 768 TFT with LED back light Touch Screen A Pin resistive with controller 10 4 inch format Processor Intel Core i3 Memory RAM Memory 4 GB DDR2 Hard drive size 16 or 32 GB Mini PC13 BIOS 2 MB flash Operating System Windows embedded system External Connectors 2 USB 2 0 1 Ethernet 1 Power input plug Power Input 12 VDC 7 5 Amps External Button On off 2 color button Orange Standby Blue Run Circulation and Cooling Allow at least 2 inches of unobstructed air circulation around the vent holes on the Auto 1000 while in use to ensure adequate cooling of the product Getting Support We provide free consulting for your lab on cell type assay type and sample conditions The goal is to find the most suitable cell counting analysis solution for your lab Our team of Application Specialists are trained biologists with comprehensive understandings of cell counting viability assay methods and best practices Please contact us when you need to discuss specific types of cells sample conditions and applications We are available between 8 30am and 5 00pm Eastern US time For immediate
15. t area of the cell image is currently displayed Pan Moves the area currently being shown within the current cell image Zoom Zooms in or out of the current cell image Count Starts counting of the current cell image Abort Preview Cancels current cell preview and returns Assay Selection Screen Current Image Select location on slide to preview CON WO OB ON N O 2013 Nexcelom Bioscience LLC Counting Screen Current Count Stop Counting Current Assay Indicates which Assay is currently selected to analyze a cell sample Assays amp Settings Select to return to Assay Selection Screen disabled while count is in progress Sample ID Select to enter a Sample ID disabled while count is in progress Dilution Factor Select to enter a Dilution Factor disabled while count is in progress Current Count Shows cell count numbers Counting Progress Visually indicates the current count progress Image Being Counted Indicates which image Is currently being counted Stop Stops cell counting O N WO OB ON 10 2013 Nexcelom Bioscience LLC Results Screen Assay Cell line total cell conc Sample ID Cell Line Dilution Factor 1 00 Total Count Concentration Mean Diameter 612 cells 1 09x106 cells ml 15 5 microns AOE View Details of Calculate and Adjust Send Results to Printer Begin Next Sample Counting Results Current Assay Indicates which Assay is currently selected
16. to analyze a cell sample Assays amp Settings Select to return to Assay Selection Screen Sample ID Select to enter a Sample ID Dilution Factor Select to enter a Dilution Factor Results Displays cell counting results including total cell count live dead cell count and viability Details Select to go to the Details Screen Sample Adjustment Calculator Select to launch the sample adjustment calculator Useful for sample adjustment to get desired concentration or total cell number 8 Print Send the counting results to a network printer 9 Done Select to start a preview of a new cell sample using the same Assay settings NO On BP WD 2013 Nexcelom Bioscience LLC 1 Calculate and Adjust Sample Screen umber of Cells MS 4 09x108 Cells ml Total Cell Number in Sam 1 09x105 Cells Sample Adjustment pees 1 84 ml of sample ip me 50 Back to Results 1 Sample Adjust Source Select which concentration source to use for the Sample Adjustment calculations 2 Original Sample Volume Select to indicate the original sample volume of the cell sample 3 Target Select to indicate the target concentration or number of cells which should be calculated 2013 Nexcelom Bioscience LLC Details Screen save Copy of Data View Gombined Image oom Inuit Go Back to Results select Image to View Current Assay Indicates which Assay is currently selected to analyze a cell sample
17. ultured primary cells without d bris Preview Image for Current Assay oad Image Edit Instrument Access User Help Settings Current Assay Indicates which Assay is currently selected to analyze a cell sample Edit Assay Select to edit the Assay and Cell Type settings Import Assay Select to Import additional assays Sample ID Select to enter a Sample ID Dilution Factor Select to enter a Dilution Factor Assays Available List of Assays available for selection Preview Select to Preview the cell sample image Load Select to load and analyze previously saved images Settings Select to edit instrument and UI settings Help Select to access help options Left Right Arrows Select to page left right to access all assays available SOON O O BB NN oo 8 2013 Nexcelom Bioscience LLC Preview Screen Current View Coarse or Fine Focus Nr A Bnghter Darker Zoom In Out pr e 12 Count Current Image Return to Assays select image to View Current Assay Indicates which Assay is currently selected to analyze a cell sample Assays amp Settings Select to return to Assay Selection Screen Sample ID Select to enter a Sample ID Dilution Factor Select to enter a Dilution Factor Focus Fine and coarse focus adjustment of the current cell image Exposure Adjust the exposure time of the current cell image Current View Red rectangle to show wha

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