Cellometer Auto M10, Cellometer Auto T4 & Cellometer Auto T4 Plus User Manual © 2010 Nexcelom Bioscience LLC
The Cellometer Auto is a high precision imaging instrument. If you have difficulty concerning operation or installation, please contact Customer Service at Nexcelom Bioscience LLC. Do not attempt to service this instrument yourself. The warranty registration must be completed and returned to Nexcelom Bioscience LLC within fourteen (14) days of purchase. Customer Service Nexcelom Bioscience LLC 360 Merrimack St. Bldg. 9 Lawrence, MA 01843 www.nexcelom.
Note: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications.
Table of Contents Part I Introduction and Description 8 1 Cellometer counting ................................................................................................................................... chamber 9 2 9 Cell counting................................................................................................................................... steps 3 ................................................................................................................................
Cellometer Auto M10, Cellometer Auto T4 & Cellometer Auto T4 Plus User Manual 7 Count from ................................................................................................................................... saved images 38 8 Save images................................................................................................................................... 38 9 .....................................................................................................................
Introduction and Description
Introduction and Description Introduction and Description 8 © 2010 Nexcelom Bioscience LLC
1 Introduction and Description The basic principle of the Cellometer® automatic cell counter is imaging cytometry. It is the same as hemacytometer in terms of spreading cells into a thin layer of liquid, which is done in a disposable counting chamber. Cellometer automates the cell counting by taking images of cells in the counting chamber, analyzing the number of cells in each image using imaging analysis, and converting the number of cells into cell concentration per ml.
Getting Started
Getting Started Getting Started 12 © 2010 Nexcelom Bioscience LLC
2 Getting Started This section helps you to get started using the Cellometer Auto systems for cell counting. 2.1 Installation This section goes through the complete procedure of installation of Cellometer Auto Counter. The instrument should be unpacked and inspected for mechanical damage upon receipt. Mechanical inspection involves checking for signs of physical damage such as: broken knobs, etc. If damage is apparent contact dealer or Nexcelom Bioscience LLC.
Getting Started Installation procedure A. Check computer system The computer operating system (Windows 2000, XP, Vista, 7 or higher), USB 2.0 port, user account with administrator privileges are essential for the installation of Cellometer Auto Counter software. B. Cellometer Auto Counter software installation 1. Insert Cellometer Auto Counter software disk into CD drive (the following images are screenshots using Windows XP, these may appear slightly different depending on the version you are using.) 2.
Installation 3.
Getting Started 4. Click "Finish" 5. The Cellomter Auto Counter icon will appear on your desktop and is ready for launching the Cellometer Auto Counter software. 6. Remove disk from CD drive. C. USB driver software installation 1. Connect power to Cellometer Auto instrument and turn on power switch located on the back of the unit. 2. Connect the USB cable between Cellometer Auto instrument and a USB 2.0 port on the computer.
Installation 3. Select the option "Yes, this time only" 4. Click “Next” 5. Select “Install the software automatically (Recommended)” 6.
Getting Started 7. Click “Continue Anyway” 8. Click “Finish” Note: some computer systems may require USB driver installation for a second time. If so, repeat steps 3 through 7, if prompted. A background image is a blank image of the system itself without the counting chamber. It is used to normalize the cell counter and is taken at the time of system installation. Unless the instrument is moved, there is generally no need to take a background image before cell counting. 1.
Installation 2. Enter instrument serial number which is located on the back of the Cellometer. 3. You will see a warning that a background image must be taken if this the first time the software is run. Click OK. 4. From the Options menu, select “Take Background Image”. 5. If necessary, remove the counting chamber from the instrument, then press OK to proceed. 6. After background image has been taken click © 2010 Nexcelom Bioscience LLC , and verify a uniform gray image.
Getting Started 2.2 Set up Cellometer Auto for cell counting This section goes through the complete procedure of counting a cell sample. You need to prepare the following sample. 1. Cell sample concentration range corresponding to instrument: • Auto T4: 2.5 x 105 to 1.0 x 107 cells/mL • Auto M10: 5x105 to 2.0 x 107 cells/mL 2. 0.2% trypan blue when viable cell count is required (0.2% trypan blue is made by diluting 0.4% trypan blue with PBS prior to use). 1.
Set up Cellometer Auto for cell counting Hold the loaded chamber in the white area and stay away from the clear optical window area. Insert the loaded chamber into Cellometer Auto sample slot, and gently push the slide to the stop. The instrument will count and analyze only the chamber inside the system. To measure the second chamber, remove the slide from Cellometer Auto, turn around the slide, and insert the second chamber in the instrument.
Getting Started Click "Count" button while the cell image is displayed. ("Display Image" button must be pressed first to enable "Count" button.) The progress bar at the bottom of the software panel indicates counting in progress. A numeric counter also displays updated cell counts. Cell counting data are displayed in the left section of the software panel. Cell count, mean diameter, and cell concentration are displayed.
Software Features
Software Features Software Features 24 © 2010 Nexcelom Bioscience LLC
3 Software Features This section describes software features of Cellometer Auto Counter software. 3.
Software Features 24) # of live and dead cells: + or - number of live or dead cells for manual adjustment 25) Manual Adjustment Enter: adjust number of cells based on manual inspection 26) Get Help button: direct access to user manual, on-line resources, video tutorials and contact with Applications Specialist. Requires internet connection.
Cellometer software panel 3.2 Cellometer software menu items Load Image for Display Load one image from saved Cellometer Auto images for display only Load Image and Count Load two saved Cellometer Auto images for counting. One Cellometer Auto image can be used twice. New Data File Change to a different data file or name a new data file where Cellometer Auto counting data is saved.
Software Features Set Current as Default Set Cell Type currently selected in the Set Up box as the Cell type after restart Cellometer Auto Counter software. 1. Cell type name 2. Cell type description 3. Minimum cell diameter that is counted 4. Maximum cell diameter that is counted 5. Roundness of the cells that are counted 6. Contrast of cells counted 7. Define degree of dark center for dead cell count 8. Define uniformity with cell for dead cell count 9. Check to pick up very dim dead cells 10.
Cellometer software menu items “Counting Options” Count All - count all cells in total areas Speed Count - stop counting after reaching user defined number of cells or images have been counted Stop Counting Over - upper limit of cell number for Speed Count Intelligent File Load - Automatically load all saved images in a file for a given sample Show Dead Cell Count- Allows you to view all of the dead cell in your sample.
Software Features "Save Options" Set sample ID as cell type: Checking this box will auto input the Sample ID to match the Cell Type parameter name being used for counting. Time stamp sample ID: Checking this box will auto append the Sample ID with the date and time the count was performed. Auto increment sample ID: Checking this box will auto append the Sample ID with an incremental numerical value.
Cellometer software menu items Save raw images / Save counted images: Checking these boxes indicate which type of images will be saved when the Auto save sample image box is checked. Auto export sample to Excel: Checking this box will auto export the counting data, results and calculations into an Excel file which will be saved in the location specified by using the Set Folder command.
Software Features Show Size Distribution Print: print cell size histogram Save As: save cell diameter values to a data file Open Diameter File: open an existing cell diameter file Multiple: check to load and display multiple cell diameter files (up to 5) Copy to Clipboard: copy diameter data and histogram to Clipboard.
Cellometer software menu items This menu item includes the following functions. Help Contents The online help consists of instructions for operating Cellometer Auto Counter and software references. Contact Support and Training Launches Nexcelom’s online resources page, which contains: video tutorials, submit a support ticket with sample images, and access to FAQs and Application notes.
Operation Reference
Operation Reference Operation Reference 36 © 2010 Nexcelom Bioscience LLC
4 Operation Reference This section describes recommended procedures to perform some routine operations with Cellometer Auto Counter software. 4.1 Background image Use the following steps for this function. 1. Place a Cellometer chamber with cells into Cellometer Auto instrument 2. “Display Image” 3. Focus if necessary 4. Remove Cellometer chamber from Cellometer Auto instrument 5. Options -> Take Background Image 6. Wait until the Background Image is saved 7. Click OK 4.
Operation Reference 8. Cell Type -> Edit Cell Type to modify cell type parameters. 9. Recount the same images using edited cell type parameter 10. Repeat steps 6 through 9 when necessary 4.5 Speed Count Use the following steps for this function. 1. Options -> Counting Options -> Speed Count 2. Input the number above which count will stop 3. Counting stops at the frame after the upper number is reached 4. Input the number of images 5. The counting stops after the upper image limit is reached 4.
Multiple sample histogram 4.9 Multiple sample histogram Use the following steps for this function. 1. Analysis -> Cell size analysis 2. Check "Multiple" 3. Open Diameter File Open first saved cell diameter file Open second saved cell diameter file .... (up to 5 diameter files) 4. Histogram of multiple sample is ready to be copied on clipboard 4.10 Copy cell size data and histogram Use the following steps for this function. 1. Open Word or Excel file 2. Analysis -> Cell size analysis 3.
Tutorial
Tutorial Tutorial 42 © 2010 Nexcelom Bioscience LLC
5 Tutorial This section includes several tutorials to illustrate how to perform some routine functions with Cellometer Auto Counter. 5.1 Cell concentration range Cell sample concentration range corresponding to the instrument: • Auto T4: 2.5 x 105 to 1.0 x 107 cells/mL • Auto M10: 5.0 x 105 to 2.0 x 107 cells/mL If the cell concentration is too high, counting results are not reliable due to clumping of cells.
Tutorial 5. Enter in dilution factor 6. Count cells 7. Output displays "Live Cell Concentration" (cells/ml), and "Viability" (%) 8. Check "Counted" Live cells are indicated by green outlines, and dead cells are indicated by red outlines, as seen in the figure (b) below. MCF7 cell viability measurement using Auto T4: (a) MCF7 cell image and (b) counted MCF7 cells.
Test viability 5.3 Set up a new cell type Method 1: Import from Cell Library Import pre-existing cell type parameters from Cell Library installed with Cellometer Auto Counter software 1. Cell Type 2. Import Cell Type 3. Select Cell Type from Cell Type in Cell Library 4. Click “>>” 5. Click “OK” 6. Review selected Cell Type in the “Set Up” “Cell Type” drop down menu Method 2: Use New Cell Type Wizard Follow interactive instructions to set up new cell type parameters using saved or live cell images 1.
Tutorial 5.4 Adjust cell size parameters For each cell type, "Cell Diameter Max." and "Cell Diameter Min." are the maximum and minimum cell sizes respectively. Cell parameters for HT-29 cell. Arrows point to cell size parameters. When cell size parameters are not set up properly, Cellometer software displays graphical indicators. Figure below show counted cells for the same HT-29 image, using difference cell size parameters. HT-29 cell counting results measured using different cell size parameters.
Adjust cell size parameters 5.5 Adjust dead cell parameters Default settings of “Dead Cell Parameters” provide fastest counting speed for standard cell types that are stained solid dark blue by trypan blue. There are some cell types with different morphology when stained with trypan blue. Weak blue dead cell In order to pick up weak blue dead cells, activate “Very Dim Dead Cells” by check “Modify Dead Cell Parameter” and check “Very Dim Dead Cells”.
Tutorial Dead cells with a brighter center Dead cells of this type have small bright centers, as shown in the following figure for CHO cells. Dead cell parameter "Sensitivity" is modified to pick up this type of dead cell. CHO cell counting results with different "sensitivity" settings. (a). CHO cell image, (b) to (e) sensitivity of the dead cell parameter was increased incrementally from 1 to 4, respectively. Green outlines indicate live cells while red outlines indicate dead cells.
Adjust dead cell parameters 5.6 Adjust decluster parameters for clumpy cells For decluster function, there are two parameters used by Cellometer software to separate cells connected to each other. They are "Decluster Edge Factor" and Decluster threshold factor ("Decluster Th Factor"). The default settings of "Decluster Edge Factor" and "Decluster Th Factor" are 0.5 and 1.0, respectively. For the majority of cell types, default settings are adequate to separate clumpy cells.
Tutorial 5.7 Turn on and off declustering for small cells Examples of small cells are mouse splenocytes, lymphocytes, thymocytes and PBMCs. Their diameters are typically less than 7 μm. For really small cells, Cellometer Auto focus may influence cell counting when the decluster feature is activated. The following figures show mouse thymocyte counting results, (a) with decluster, and (b) without decluster. The green outlines indicate counted cells.
Turn on and off declustering for small cells However, some samples contain clumpy cells, as shown in the following figure (a). Other samples have very high cell concentration, as shown in 3.7.3 (b). For these samples, Decluster is activated, and Contrast Enhancement is reduced. Cell type parameters for high concentration or clumpy small sized cells. 5.
Tutorial Calculator #1: Desired cell concentration Input: Target Concentration Click: Apply Change Display: Instruction for dilution or concentration in Sample Adjustment Calculator #2: Target Number of Cells Input: Target Number of Cells Click: Apply Change Display: Pipetting volume and number of aliquots in Sample Adjustment 52 © 2010 Nexcelom Bioscience LLC
Sample adjustment after cell couting 5.
Tutorial Print: print cell size histogram Save As: save cell diameter values to a data file Open Diameter File: open an existing cell diameter file Multiple: check to load and display multiple cell diameter files (up to 5) Copy to Clipboard: copy diameter data and histogram to Clipboard.
Appendix
Appendix Appendix 56 © 2010 Nexcelom Bioscience LLC
6 Appendix 6.1 Spectifications Cellometer Auto is an imaging instrument that acquires cell data from multiple locations of the Cellometer counting chamber. It is connected to a computer via USB 2.0 port. Instrument Specifications Weight: 9 lb ( 4 kg) Size: 3.5 in x 4 in x 12.5 in ( 9 cm x 10 cm x 32 cm ) Wall Adaptor: 100-240 AC, 50-60 Hz, 0.5 A Power to instrument : 12 V DC, 1.5 A Max 6.
Appendix a. On your PC, go to Start->Control Panel b. Double click “System” The operating System must be Windows 2000, Windows XP (SP2 or SP3), Vista, Windows 7 or higher for Cellometer Auto Counter software to run. Installed file locations Program Files • File location for default settings on English Windows XP and Windows Vista or Windows 7: “C:\Program Files\Nexcelom\” • On installation CD: “\program files\Nexcelom\” Files: MFC42D.dll MFCO42D.dll MFCRTD.dll NexcelomIcon.
Computer check procedure \Cell Samples – Contains sample images Files: AutoT4-HEK293-A.bmp AutoT4-Hela-A.bmp AutoT4-HT29_blue-A.bmp AutoT4-Jurkat-A.bmp AutoT4-SF9_blue-A.bmp AutoT4-Vero-A.bmp \Docs Files: CellometerAutoCounter.pdf – Software manual in .pdf form \Drivers – This folder contains a copy of the AutoT4/M10 USB drivers.
Appendix USB memory stick installs It is often the case that installing from a USB memory stick will result in a failure to load USB drivers during install and crash the install. If this happens, copy the install from the memory stick to the desktop of the computer and install the software from the desktop location.
Customer Service Nexcelom Bioscience LLC 360 Merrimack Street Building 9 Lawrence, MA 01843, USA Phone: 978-327-5340 Fax: 978-327-5341 Email: support@nexcelom.com www.nexcelom.com Rev.
