Nexcelom Cellometer Auto T4 User Guide
- June 9, 2024
- Nexcelom
Table of Contents
Nexcelom Cellometer Auto T4
PRODUCT DESCRIPTION
- Display Image Button – Shows cells currently in the counting chamber.
- Count Button – Initiates counting procedure.
- Test Viability – Indicates that a viability stain has been used.
- Cell Type – Identifies cell type parameters to be used when counting.
- Sample ID – Allows entry of a unique sample identifier.
- Dilution Factor – Allows entry of final dilution factor to calculate concentration.
- Live/Dead Count – Displays live and dead cells found if Test Viability is selected.
- Cell Count or Total Cell Count – Displays total cells counted in the images taken.
- Mean Diameter (micron) – Identifies mean diameter of counted cells in microns.
- Viability (%) – Identifies percent viability of cell sample.
- Live Cell Concentration – Indicates calculated live cell concentration.
- Cell Concentration or Total Cell Concentration (Cells/mL) – Indicates calculated cell concentration/total cell count concentration.
- Recount Icon – Re-analyzes images for current scan and performs a new count.
- Select Data File Icon – Opens Windows File Explorer for saving count results
- Image Area – Displays images captured by the instrument.
- Save Data Button – Enables users to save the data in the indicated file.
- View Saved Data Button – Displays the data that would be saved.
- Calculate Concentration Adjustment Icon – Activates Sample Adjustment tool.
- Cell Size Analysis Icon – Activates Cell Size Analysis tool generating a histogram of cell size data to assist users with optimization of cell diameter parameters.
- Print Results Icon – Prints results to selected printer.
- Export to Excel Icon – Exports results to MS Excel worksheet.
- View Image – Toggles between images A and B; zooms into fields of view 1-4.
- Counted – Displays graphic overlay highlighting counted cells.
- Count in View or Live/Dead Count in View – Displays total number of cells in the currently selected view if Test Viability is selected.
- Manual Adjust or Live/Dead Manual Adjust – Enables users to manually adjust cell numbers by manually entering a count of cells in view.
- Help Icon – Provides users with access to online help contents and resources.
Contents of Shipping Container
- Cellometer Auto T4 Instrument
- Power Cord
- USB Connector Cable
- USB Drive − Contains Cellometer Auto T4 Software
- Cellometer Auto T4 User Manual and Quick Start Guide − PDF files stored on USB Drive
- Focus Guide − Graphic sheet intended to help users adjust instrument focus
- Counting Chamber Slides − One box of 75 disposable slides; two counting chambers per slide
- Nexcelom-provided Laptop (optional)
Unpacking the Instrument
Unpack and inspect the Auto T4 to ensure no damage has occurred during shipping. For details on unboxing and getting started with the instrument, visit the Cellometer Auto T4 page on our website, click the Resources tab and scroll down to find Training Videos.
Site Preparation
Instrument must be placed on a level surface and plugged directly into a surge
protector (recommended) or power outlet. Ensure all cables are free from
tangles prior to starting the Auto T4. Keep the area around the instrument
clean between, during and post operation. Do not position the device so that
it is difficult to disconnect from power main.
Operating Computer Requirements
If you did not purchase an Operating Computer from Nexcelom to run your Auto
T4 instrument, the following minimum specifications are required:
- Windows 7, 8 or 10 PC
- 2.2 GHz or higher processor
- 4 GB RAM
- USB 2.0 port or higher
- Display resolution: 1024 x 768 pixels or higher
Installing the Software
The Auto T4 System is comprised of the instrument connected via USB cable to
an Operating Computer used to run the instrument software. The Operating
Computer can be linked to a network for accessing external files, printers and
for storing count results. If the Operating Computer was purchased from
Nexcelom, software will be pre-installed and you can skip this section.
Continue to Starting the Auto T4. Ensure that instrument is not connected to
the computer during software installation until specified in the procedure.
- Insert Software USB Drive that was included with the instrument into USB port of the Operating Computer.
- Extract files contained on the USB to the computer desktop.
- On the desktop of Operating Computer, double-click the Cellometer_ version.exe file (where version represents version being installed).
- Click Next to begin the Auto T4 software installation
- Click Install to install the instrument software on the hard drive of the Operating Computer. A screen containing prerequisite software to be installed on your computer may be displayed. Click Install to continue.
- When prompted, click OK to check for prior versions of the software.
- Click Finish to exit the installer and confirm that Cellometer Auto software icon now appears on desktop of Operating Computer.
- Remove the Software USB and store it per company guidelines.
- Plug power cord for the instrument into an outlet and power ON.
- Once software installation is complete, connect USB 2.0 cable between the Auto T4 and a USB 2.0 port on the Operating Computer.
Using Instrument for the First Time
Starting the Auto T4
- On the desktop of Operating Computer, launch the Auto T4 software by double-clicking the Cellometer Auto software icon.
- Enter instrument Serial Number from the device label into the input fields. The device label can be found on the back of instrument.
- Click Done to save the instrument Serial Number.
- A pop-up message will appear indicating a background image needs to be taken before counting. Click OK to continue
Taking a Background Image
After initial setup of the instrument, you must take a background image of the
system by itself (without counting chamber slide inserted) to normalize the
cell counter. Unless the instrument is moved to a new location, there is
generally no need to take another background image.
- On the desktop of Operating Computer, launch the Auto T4 software by double-clicking the Cellometer Auto icon.
- Navigate to the Options Menu and select Take Background Image option.
- Remove slide from instrument, if applicable. Click OK in response to pop-up message to confirm Auto Sample Slot is empty.
- The system takes the background image and displays a pop-up indicating when image has been saved. Click OK.
- Click the Display Image button located in top left corner of screen.
- Confirm that background is a uniform gray color. If there is any discoloration (e.g., light/ dark areas), contact Nexcelom Support at +1 978-327-5340 or via email: [email protected]
Setting Up Auto Save Feature
- Open the Options Menu and select the Save Options… option.
- To automatically save data, click the Auto Save data.txt check box to select it. By default, count results will be saved to a single data.txt file in which rows (logged with date/time stamp and Sample ID) are appended each time data is saved. To change the location or name of this data file, click the Set Folder button, navigate to a new location, enter a file name and click Open to create the file.
- To automatically save data to a new data file each time cells are counted, click the Create new data.txt with each sample check box to select it. Keep in mind selecting this option may result in a large volume of files. Click the Set Folder button, navigate to a new location, enter a file name and click Open to create the file.
- To automatically save images, click the Auto Save Sample Image check box to select it. Indicate whether you want to:
- Save Raw Images (saved as .png files; images can be loaded back into Auto T4 for reanalysis),
- Save JPG Copies (images cannot be loaded back into Auto T4; use for adding to presentations or reports), or
- Save Counted Images (also saved as .jpg files; images include graphic overlays of outlined cells).
- Click the Set Folder button, navigate to a location on the Operating Computer or network where image files can be saved, enter a file name and click Open to return to create the file.
Preparing Sample Slides
Although the Auto T4 does not require routine testing or calibration, counting
beads are available to verify instrument functionality. Nexcelom counting
beads BO5-02-050 (5 μm), B10-02-020 (10 μm) and B15-02-010 (15 μm) are
recommended for use with Cellometer instruments.
To prepare slides containing counting beads or cell samples:
- For SD100 slides, remove protective film from both sides of the slide. PD100 slides do not have this film. Note that it may be difficult to peel the film from the slide.
- Place the slide on a fresh Kimwipe.
- To prepare two samples at once, label individual chambers (e.g., Chamber 1 and Chamber 2) in the white margin.
- Take care to ensure that the clear optical windows of the counting chamber are not touched.
- Invert counting bead solution or tube containing cell sample a total of 10 times. If using counting beads, vortex bead solution for 10 seconds. Do not vortex cell samples.
- Set pipette to 20 μL and then pipette bead solution/cell sample up and down ten times (20x) to break up any potential clumps.
- Load 20 μL of counting beads or cell sample into counting chamber of Cellometer slide (CHT4).
Adjusting Instrument Focus
- While previewing a sample slide loaded into the instrument, adjust focus by slowly turning the Focus Knob located on the right side of the Auto T4.
- Proper focus is key to ensuring accurate cell counts.
- Counting beads will appear as dark circles with sharp edges
- Live cells should have a bright center and dark, crisp clearly defined edges.
Counting and Analysis Workflow
- In the Setup area of the Home screen, choose a cell type from the Cell Type drop-down list, enter a Sample ID and ensure Dilution Factor is accurate.
- While the cell image is displayed, click the Count button. When software is in Data Analysis Mode, click the Recount icon to re-analyze current scan and perform a new count.
- Once counting is complete, results are displayed in the bottom left and right corners of the screen.
- Use the controls in the View Image area to view different areas of counting chamber.
- Confirm data is accurate by clicking the Counted check box to view image using a graphic overlay outlining counted cell
- If testing for cell viability, select the Test Viability check box in the Setup area.
- Additional count results including Viability (%) are displayed.
- Click the Counted check box to view image using graphic overlay outlining counted cells
Cell Size Analysis Tool
The Cell Size Analysis tool displays cell size data from count results by
generating a histogram that enables users to optimize cell diameter parameters
for the sample (e.g., to exclude debris or very large cells) and refine count
data.
Sample Adjustment Tool
The Sample Adjustment tool assists you in calculating the adjustment
necessary to achieve a specified target concentration or target number of
cells in a tube.
Using Trypan Blue Viability Method
When evaluating viability methods, it is critically important to use a single
aliquot from the stock cell culture to perform all testing. The cell sample
should be evaluated for concentration on the Auto T4 prior to staining.
Brightfield imaging used in conjunction with Trypan Blue Viability may be used
to determine the number, concentration and percentage of live cells for cell
lines and cultured primary cells. Brightfield imaging with trypan blue
viability is not recommended for samples containing debris, platelets or red
blood cells. In these cases, fluorescence is required to accurately
differentiate nucleated cells from platelets, red blood cells and debris.
Preparing Sample for Trypan Blue Viability Determination
Cell concentrations of 1.0 x 105 − 1.0 x 107 cells/mL can be analyzed on the
Auto T4, with a concentration of 1.0 x 106 cells/mL being optimal. Invert the
tube containing cells ten times (10x) and pipette up and down 10x to generate
a homogeneous cell sample and reduce cell clumps. Do not shake or vortex the
sample as this may damage cell membranes. For viability measurement, stain
cells by combining 50 μL of cell sample with 50 μL of a 0.2% trypan blue
staining solution (for a final concentration of 0.1% trypan blue). Gently mix
by pipetting up and down 10x
Cell Concentration and Trypan Blue Viability
- Pipette up and down gently ten times (10x) to break up any potential cell clumps in the stained cell sample.
- Load 20 μL of sample into the Cellometer counting chamber (CHT4).
- Hold the loaded chamber in the white area and insert counting chamber containing sample into Auto Sample Slot until touching the internal stop.
- Click the Display Image button in the top left corner of screen and adjust instrument focus using knob on side of instrument.
- Click the Test Viability check box located in Setup section of left panel.
- Click the Count button to perform the count.
- When counting is complete, results for Live/Dead Cell Count, Total Cell Count, Mean Diameter (micron), Viability (%), Live Cell Concentration and Total Cell Concentration are displayed in bottom left corner of screen.
- Review counted image by clicking the Counted check box located in the View Image section of right panel.
- If Auto Save feature has not been enabled, save your data (if desired). The Auto T4 is now ready to analyze the next sample.
Contacting Nexcelom Support
Nexcelom Support is available from 8:30 am to 5:30 pm EST and can be reached
at +1 978-327-5340 or by sending email:
[email protected] Trained specialists are available
to assist with sample analysis and optimization of assay/cell type imaging
parameters.
See the following documentation for additional instrument information:
- 8001009 Cellometer Auto T4 User Manual for instrument operation, care and maintenance details (available as a PDF file on the USB Drive)
- 8001197 Cellometer Auto T4 Focus Guide for assistance with optimal focus