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Automated cell counter
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Luna-FL™ Automated Cell Counter
User Manual
Catalog No. : L20001
Document No. : L24001
Revision Date : July 15, 2013
Version : 1.0.0
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  • Page 1 Luna-FL™ Automated Cell Counter User Manual Catalog No. : L20001 Document No. : L24001 Revision Date : July 15, 2013 Version : 1.0.0...

  • Page 2: Table Of Contents

    IMPORTANT Luna-FL™ Automated Cell Counter is a Laboratory Electrical Instrument for Scientific Research Use Only. It is NOT A MEDICAL or IN VITRO DIAGNOTICS DEVICE. Table of Contents Safety Information -------------------------------------------------------------------------------------------- General Guidelines ------------------------------------------------------------------------------------------- Environment Conditions ------------------------------------------------------------------------------------- Chapter 1 – Introduction --------------------------------------------------------------------------- 1.1 Product Overview -------------------------------------------------------------------------------------...
  • Page 3 Chapter 4 – Fluorescence Cell Counting ------------------------------------------------- 4.1 Sample Preparation ---------------------------------------------------------------------------------- 4.2 Loading Samples into Slides ----------------------------------------------------------------------- 4.3 Counting Cells ----------------------------------------------------------------------------------------- 4.4 Using the “Tag” Function ---------------------------------------------------------------------------- 4.5 Cell Size and Number Distribution --------------------------------------------------------------- 4.6 Using the Dilution Calculator ----------------------------------------------------------------------- 4.7 Saving Images and Generating a PDF Report of the Current Count -------------------- 4.8 Printing the Counting Results ---------------------------------------------------------------------- Chapter 5 –...

  • Page 4: Safety Information

    Safety Information For the best results, users of the Luna-FL™ automated cell counter (or, hereinafter “Luna-FL™”) must follow the instructions below in addition to the general precautions for using electrical instruments. 1. Users must be careful to avoid electric shock while operating the instrument. Do not touch it or its components with wet hands.
  • Page 5 <Symbols used in this User Manual> The WEEE (Waste Electrical and Electronic Equipment) symbol indicates that users of this instrument have the responsibility of returning and disposing of WEEE in an ecologically friendly manner. Follow waste ordinances of your region for proper disposal provisions.

  • Page 6: General Guidelines

    General Guidelines for Using the Luna-FL™ Automated Cell Counter In order to achieve the best results with the Luna-FL™ automated cell counter, users must follow the instructions below carefully. The instrument must be operated in compliance with the environmental conditions described on page 7.

  • Page 7: Environment Conditions

    Environmental Conditions Operating Power 100~240 VAC, 1.5A Frequency 50/60 Hz Electrical Input 12 VDC, 3.5A Installation Site Indoor use only Operating Temperature 10~35℃ Maximum Relative Humidity 20~80% Altitude ≤2,000 m Pollution Degree...

  • Page 8: Chapter 1 - Introduction

    The Luna-FL™ helps measure the number as well as the viability of cells (live, dead, total cells) with sophisticated optical components and advanced image analysis algorithms. Due to the innovations introduced by Logos Biosystems, the Luna-FL™...
  • Page 9 Histogram of cell size distribution and size gating The Luna-L™ cell counting chamber slide is disposable and specifically designed for the Luna-FL™. Each counting slide has two chambers, labeled A and B, one slide can be used for the same sample reading in duplicate or it can be used for two different samples if preferred.
  • Page 10 Each user can set or adjust counting parameters and save the Individual protocol saving counting protocol for later use. Luna-FL™ cell counter provides a counting report based on Automatic reporting the count results in PDF (Portable Document Format) immediately after performing a cell count.

  • Page 11: Product Contents

    1.2 Product Contents The Luna-FL™ automated cell counter includes following components. Component Quantity Luna-FL™ automated cell counter 1 unit Power cord (including an adapter) 1 unit Luna™ cell counting chamber slides 1 box of 50 slides AO/PI cell viability kit 2 X 0.5 ml...

  • Page 12: Product Specifications

    Processing Time Approx. 7 seconds for bright field & approx. 30 seconds for dual fluorescence (may depend on cell type and concentration) 1.3.2 Luna-FL™ cell counter counting slide specifications Dimensions (WxDxH) 25 mm X 75 mm X 2.4 mm Chamber Depth 100 m...

  • Page 13: Product Description

    1.4.2 Rear view of the Luna-FL™ automated cell counter The rear view of the Luna-FL™ automated cell counter shows a power button to turn on or off the instrument and a power inlet. Connect the instrument to an electrical outlet with the power cord and plug provided in the product package.
  • Page 14 1.4.3 Right side view of the Luna-FL™ automated cell counter The right side view of the Luna™ automated cell counter shows a focus knob and a slide port. Use the focus knob to get optimized cell images by adjusting contrast between live cells with bright centers and dead cells with dark centers.
  • Page 15 1.4.5 Luna-FL™ cell counting chamber slide The plastic disposable cell counting chamber slide consists of two chambers, labeled as A and B, that can be used for the same sample as duplicates or for two different samples. The depth of the counting chamber is 100 m.

  • Page 16: Chapter 2 - Setting Up

    2.1.1 Upon receiving the product package, unpack it carefully and ensure that every component is included and no damage has occurred. 2.1.2 Place the Luna-FL™ automated cell counter on a flat, stable surface. 2.1.3. Insert one end of the power cord into the instrument and plug the other end of the power cord into an electrical outlet after checking the outlet configuration in your local area.

  • Page 17: Start-Up Screen

    2.2 Start-Up Screen 2.2.1 Home Screen When the Luna-FL™ is turned on, the Start-Up screen will display two options: one for the Bright Field Cell Counting and the other for Dual Fluorescence Cell Counting. Users can choose one of them.
  • Page 18 2.2.3 Fluorescence Counting Screen 2.2.3.1 Green Fluorescence Counting Screen 2.2.3.2 Red Fluorescence Counting Screen...

  • Page 19: Settings

    2.3 Settings Generally, users do not need to change the “Settings” of the instrument since they are preset at the time of manufacture. If users need to reset the date, time, or other options, users can adjust or change the options/parameters from the “Settings” menu described below. 2.3.1 After turning on the instrument, go to one of the counting screen options.

  • Page 20: Setting The Date And Time

    2.4 Setting the Date and Time 2.4.1 On the “Settings” screen, select the date and time buttons. 2.4.2 After selecting the field, erase the number using the “Backspace” button ( ), set the date or time using the number buttons, and press the “Apply” button ( ) to save the changes.

  • Page 21: Counting Options

    “Auto-header” in the root directory of the USB drive. In case users want to have another default name, the “Auto-header” can be customized by editing the default value of “Logos Biosystems-”. 2.6 Counting Options 2.6.1 The “Counting Options”...
  • Page 22 Without trypan blue When samples do not contain trypan blue dye, turn on this button and follow the directions in the message boxes. These instructions set the “Dilution Factor”. For samples with trypan blue dye, the dilution factor must be set to value “2”. For samples without trypan blue dye, the dilution factor must be set to value “1”.

  • Page 23: Result/Printer/Calculator Options

    2.7 Result/Printer/Calculator Options 2.7.1 This option shows three different menus, as below. <Result/Printer/Calculator Options> Option Description Result The default value is “off.” When it is turned on, the counting result box displays only the total cell number. Dilution Calculator The default value is “Total Cells (on).” In order to use the “live cells” in the Dilution Calculator, please turn the “live cells”...

  • Page 24: Chapter 3 - Bright Field Counting Cells

    3.1.3 Mix 10 l of the cell sample with 10 l of trypan blue stain gently in a microfuge tube by pipetting up and down. Note: The cell counting parameters of the “Bright Field Cell Counting” mode of Luna-FL optimized with the use of trypan blue. Although the counting “Without Trypan Blue” option is provided in the protocol menu (section 2.6.2) to meet the various needs of users, it is NOT...

  • Page 25: Counting Cells

    The instrument analyzes only the first inserted chamber. Gently insert the counting slide to the end. Note: After inserting the slide, Luna-FL™ only reads the first chamber. To read the second chamber, it must be taken out, rotated, and inserted again. Make sure that the counting slide is not inserted upside-down.
  • Page 26 3.3.3 The “Preview screen” will appear as shown in the image below. When necessary, adjust the focus with the focus knob on the right side of the instrument to make sure that live cells have bright centers and dark edges, and dead cells have uniform dark color all over them. <Preview Screen>...
  • Page 27 <Wrong Staining Option Selected: Too Dark Preview Image> Note: Default preview image is set to a grey scale. To change the preview images to a color mode, press the “Color” button once, and the preview image will turn to a color image and the “Color button”...
  • Page 28 <Color Preview Screen> 3.3.4 Press the “Count” button, which is located on top of the “Zoom-in” button. 3.3.5 Within approximately 7 seconds (depending on cell concentration), the image of the cell sample and data/results (Total, Live, and Dead cell concentrations, Viability, Avg. Cell Size, Actual number of Total, Live and Dead cells) will be displayed on the screen.

  • Page 29: Using The "Tag" Function

    Green circle – live cell Red circle – dead cell Note: This “Tag” function is one of the distinct tools of the Luna-FL™ automated cell counter because it allows the user to review data to determine the accuracy of the counting on-site without depending on a computer.

  • Page 30: Cell Size And Number Distribution

    3.5 Cell Size and Number Distribution 3.5.1 To obtain more data on the distribution of cell size and number in graphical representation, press the “Graph/Gating” button on the lower left side of the screen. A histogram will appear with more details on the size and number of the cell sample as shown below. Live cells are displayed as a green bar and dead cells are displayed as a red bar.
  • Page 31 <Combination: Live Cells and Cell Number> The combination of “Live” and “Cell number” shows the distribution of cell size vs. cell number for only live cells. <Combination: Dead Cells and Cell Number> The combination of “Dead” and “Cell number” shows the distribution of cell size vs.
  • Page 32 <Combination: Total Cells and Concentration> The combination of “Total” vs. “Cell concentration” shows the distribution of cell size vs. cell concentration for total cells. <Combination: Live Cells and Concentration> The combination of “Live” vs. “Cell concentration” shows the distribution of cell size vs. cell concentration for only live cells.
  • Page 33 3.5.1.1 Graphical representation of live and dead cells When the “Total” button icon is clicked, the histogram is expressed as a “ Stacked Column Chart.” <Graphical Representation of Live and Dead Cells> Live cells Dead cells 3.5.2 “Cluster Map” button 3.5.2.1 When the “Cluster Map”...

  • Page 34: Using The Dilution Calculator

    3.6 Using the Dilution Calculator 3.6.1 The Luna-FL™ automated cell counter provides a built-in dilution calculator that helps easily calculate adjustments to obtain a desired concentration. <Built-in Dilution Calculator> 3.6.2 The dilution calculator initially shows the current concentration. Put the appropriate numbers into the blanks of the “Desired Concentration”...
  • Page 35 Users can also calculate their custom dilution. 3.6.3 Press the “Calculate” button and then a dilution instruction will appear in the message box.

  • Page 36: Saving Images And Generating A Pdf Report Of The Current Count

    3.7 Saving Images and Generating a PDF Report of the Current Count 3.7.1 To save data and/or generate a report, insert a USB drive into the USB port on the left side of the instrument. 3.7.2 Press the “Save” button located on the lower left part of the screen and a new “Save” window will pop up on the screen as shown below.
  • Page 37 The saved files can be opened after transferring the images/report via USB drive to a PC with the appropriate software. Note: With the Luna-FL™ software, additional graphical representations of the data and results are provided in the PDF report, as shown below.

  • Page 38: Printing The Counting Results

    Note: For more information on the Luna™ Printer, please see the User Manual of the Luna™ Printer. 3.8.2 First, connect the Luna Printer to the Luna-FL™ via the supplied USB connector (The Luna Printer can be connected at any time.) Then turn on the Luna Printer by pressing the “Power” button for 1~2 sec (To turn off, press the “Power”...
  • Page 39 <Print Button> 3.8.4 The counting results and its protocol will be printed out as below. <Printed Results> Note: The “Print” button prints only the latest counting results.
  • Page 40 Note: If the “Protocol” part of the print is not necessary, users can turn off the option in the “Results/Printer/Calculator” Option in the “Settings” menu, as shown below. <Result/Printer/Calculator Options>...

  • Page 41: Chapter 4 - Fluorescence Cell Counting

    Chapter 4 – Fluorescence Cell Counting 4.1 Sample Preparation 4.1.1 Prepare the following materials to count cells  Cell sample  Luna™ cell counting slides (cat# L12001, L12002, L12003 or L12004)  AO/PI cell viability kit (cat# F23001) 4.1.2 If desired, insert a USB drive to save data and results, 4.1.3 Transfer 2 l of the AO/PI staining solution to a new 1.5 ml microfuge tube.

  • Page 42: Counting Cells

    <Slide Inserting> 4.2.2 The remaining 10 l of the sample can be used for another counting to average the counting results. Note: Be careful not to over-load or under-load the sample into the chamber. 4.3 Counting Cells 4.3.1 Wait for about 1 min in order for the cells to settle down. Note: Skipping this step will result in poor counting accuracy.
  • Page 43 The instrument analyzes only the first inserted chamber. Gently insert the counting slide to the end. Note: After inserting the slide, Luna-FL only reads the first chamber. To read the second chamber, it must be taken out, rotated, and inserted again.
  • Page 44 Note: After this step, the real image of the cell sample will be displayed on the screen. The cell image can be navigated by touching the screen and moving the finger or stylus pen on the image. Please note that the touch screen of the Luna-FL automated cell counter is a resistive touch screen, which needs a bit of pressure in order to get a response.
  • Page 45 under the “Count” button. Initially, the “Zoom-in” button is set to a 1x image. When pressed once, a 2x image appears. This 2X image may be the best zoom-in status for focusing. When pressed again, a 4x image will be shown. Pressing the button again takes the screen back to a 1x image. Note: Check if all cells in the preview screen are immobile.
  • Page 46 4.3.6 Press “BF/GF/RF selection button” one more time to preview the red fluorescence (RF) image. Adjust RF intensity value as needed. The factory set value for mammalian cells is 5. Note: During the preview step, if cells are irradiated for a long period of time, their fluorescent intensities will quickly decrease.
  • Page 47 <Fluorescence Cell Counting Results>...

  • Page 48: Using The "Tag" Function

    4.4 Using the “Tag” Function 4.4.1 To verify counting results after performing the counting function, press “Images” and “Tag” buttons as shown below. Press Images button <Tagged Cell Image> Press Tag button Green circle – live cell Red circle – dead cell...
  • Page 49 Note: This “Tag” function is one of the distinct tools of the Luna-FL™ automated cell counter because it helps users review and determine the accuracy of the counting on-site without depending on a computer. 4.4.2 When touching the “Tag” button, the image on the screen will show objects (mostly cells) surrounded by green or red circles.

  • Page 50: Cell Size And Number Distribution

    4.5 Cell Size and Number Distribution 4.5.1 To obtain more data on the distribution of cell size and number in graphical representation, press the “Graph/Gating” button on the lower left side of the screen. A histogram will appear with more details on the size and number of the cell sample as shown below. Live cells are displayed as a green bar and dead cells are displayed as a red bar.
  • Page 51 <Combination: Live Cells and Cell Number> The combination of “Live” and “Cell number” shows the distribution of cell size vs. cell number for only live cells. <Combination: Dead Cells and Cell Number> The combination of “Dead” and “Cell number” shows the distribution of cell size vs.
  • Page 52 <Combination: Total and Concentration> The combination of “Total” vs. “Cell concentration” shows the distribution of cell size vs. cell concentration for total cells. <Combination: Live and Concentration> The combination of “Live” vs. “Cell concentration” shows the distribution of cell size vs. cell concentration for only live cells.
  • Page 53 4.5.1.1 Graphical representation of live and dead cells When the “Total” button icon, the histogram is expressed as a “ Stacked Column Chart.” <Graphical representation of live and dead cells> Live cells Dead cells 4.5.2 “Cluster Map” button 4.5.2.1 When the “Cluster Map” button is pressed, the percentage distribution of the clustered cells are displayed.

  • Page 54: Using The Dilution Calculator

    4.6 Using the Dilution Calculator 4.6.1 The Luna-FL™ automated cell counter provides a built-in dilution calculator that helps easily calculate adjustments to obtain a desired concentration. <A Built-in Dilution Calculator> 4.6.2 The dilution calculator initially shows the current concentration. Put the appropriate numbers into the blanks of the “Desired Concentration”...
  • Page 55 Users can also calculate their custom dilution. 4.6.3 Press the “Calculate” button and then a dilution instruction will appear in the message box.

  • Page 56: Saving Images And Generating A Pdf Report Of The Current Count

    4.7 Saving the Image and Generating a Report of the Current Count 4.7.1 To save data and/or generate a report, insert a USB drive into the USB port on the left side of the instrument. 4.7.2 Press the “Save” button located on the lower left part of the screen and then a new “Save” window will pop up on the screen as shown below.
  • Page 57 USB drive. Now, the saved files can be opened after transferring them via USB drive to a PC with the appropriate software. Note: With the Luna-FL software, additional graphical representations of the data and results are provided in the PDF report, as shown below.

  • Page 58: Printing The Counting Results

    4.8 Printing the Counting Results Please see Section 3.8 (Page 38) to print the counting results.

  • Page 59: Chapter 5 - Reviewing Saved Count Results

    Chapter 5 – Reviewing Saved Count Results The Luna-FL™ automated cell counter provides an easy-to-use review function using the previous count results saved in the USB drive for further review. 5.1 Importing Previous Counts 5.1.1 Insert the USB drive containing the previous results into the USB port of the instrument.

  • Page 60: Reviewing Previous Results

    5.2 Review previous results 5.2.1 Press the file name, and the previous result will start to be imported. Both the previous image and the counting results will be displayed onto the instrument screen. <Reviewing the previous result> 5.2.2 The previous image can be viewed at 1X, 2X or 4X by pressing the “Zoom-in” button and navigated by touching and dragging the image window.

  • Page 61: Previous Count" Button

    5.3.1 Pressing the “Previous Count” button on the left side of the “Review” menu will bring up a new window. The “Previous Count” window contains up to 1,000 previous counting results that have been saved in the memory of Luna-FL. <”Previous Count” Button>...
  • Page 62 In the window of the “Previous Count,” there are 3 buttons: 1. “Export to USB (.CSV)” – Users can save the data of all previous counts into a USB memory drive, which can be handled in PC software. 2. “Erase all” – All data can be removed from the memory. 3.

  • Page 63: Chapter 6 - Setting Up The Protocol For Counting

    Chapter 6 – Setting Up the Protocol for Counting Users can have unique protocols for each and apply them to specific cell types. When the “Protocol” button is pressed on the main screen, a list of protocols will show up in the protocol list box. Note: Initially, there are two protocols named as “DEFAULT”...
  • Page 64 The adjustable parameters are described as follow: Parameter Range Default Description The default value for the dilution factor is “2”. Users can modify this value according to the dilution of the original samples. If diluted for “A” times, users set this variable to “2xA”.

  • Page 65: Adjusting Parameters For Fluorescence Counting

    cell types that are not generally circular or are irregularly shaped. Minimum Use this parameter to set the minimum cell size for 1-90 Cell Size inclusion. The unit is 1 micrometer. Maximum Use this parameter to set the maximum cell size for 1~90 Cell Size inclusion.

  • Page 66: Managing Protocols

    (8 l), 2.00 (10 l), 2.50 (12 l), 3.33 (14 l), 5.00 (16 l) to 10.00 (18 l). Dilution factor corrects the actual number of cells; thus the analyzed results are generated for the original samples. Dilution factor will help the users during repeated counting of the samples of high cell density such as fermented CHO cells.
  • Page 67 After adjusting the parameters, press this button to save a new protocol. Save as After saving, users can load the protocol for future counting. Note: The Luna-FL automated cell counter is provided with a factory setting protocol named “DEFAULT”. This “DEFAULT” protocol cannot be edited or deleted. The parameters in the “DEFAULT”...

  • Page 68: Chapter 7 - Maintenance And Troubleshooting

    Luna- FL™ cell counter. Be sure to turn off the Luna-FL™ cell counter and disconnect the power cable before cleaning and performing any other maintenance. Ensure that water and other solutions do not enter any part of the instrument during cleaning.

  • Page 69: Changing The Battery

    7.2 Changing the Battery The battery of the Luna-FL™ automated cell counter is expected to last for 10 years. However, if the date/time changes or slows with unknown causes, it may indicate that the battery is weakening or running out. To change the battery, contact service personnel of the instrument supplier in your country.

  • Page 70: Calibrating The Counter

    7.3 Calibrating the Counter In general, recalibration of the Luna-FL™ automated cell counter is not necessary since it is pre- calibrated during manufacture. However, if needed for the installation or operation qualification, please follow the procedure below. 7.3.1 Turn on the instrument to show the Start-Up screen on the display. If the instrument is already turned on, please turn off and on, and then go to the “Start-Up”...
  • Page 71 1 is performed without removing the counting slide, cell counting result will be inaccurate. <Calibration Step 1> Note: Never turn off the Luna-FL™ cell counter during calibration, which could lead to a significant technical failure. 7.3.4 “Calibration Step 2” is necessary for the “Custom Staining Calibration.” From this custom calibration, users can use their own stains for cell counting.
  • Page 72 Custom Staining Calibration. To count cells under 0.1% trypan blue concentration, users can mix cells with 0.2% trypan blue solution at a 1:1 ratio. <Calibration Step 2-2> Note: Never turn off the Luna-FL™ cell counter during calibration, which could lead to a significant technical failure.
  • Page 73 Insert the counting slide and then press the “Start” button. Make sure to load the original standard solution - do not dilute it. <Calibration Step 3-1> <Calibration Step 3-2> Note: Never turn off the Luna-FL™ cell counter during calibration, which could lead to a significant technical failure.
  • Page 74 7.3.6 When the three calibration steps are done successfully, users will see the message box as below. <Successful Calibration>...

  • Page 75: Updating Firmware

    <Settings> 7.4.2 Check the firmware version and download the latest version from the Luna-FL™ website at www.logosbio.com. if necessary. Save the file in a USB drive. Note: The firmware, consisting of 1 file, must be saved into the root directory of the USB drive.

  • Page 76: Troubleshooting

    7.5 Troubleshooting Problem Possible Cause Solution Ensure that the cell image is appropriately focus by using the focus knob. You may use the “Zoom- Unfocused cell image in” button when adjusting the cell image. Make sure that live cells have bright centers and dead cells have dark/blue centers.
  • Page 77 The more cells that are counted, the Low precision range more precise the result. Although Luna-FL can count as low as 5x10 cells/ml, cell concentration higher than 2x10 cells/ml is required for 10% or lower Coefficient for variation (CV).
  • Page 78 drive. The file should be located in the root directory. Third, ensure that the USB drive is inserted correctly. Last, try the update again. If the problem continues, contact your local supplier.

  • Page 79: Chapter 8 - Ordering Information

    The following products can be ordered from your regional supplier or the website (www.logosbio.com). Cat # Product Quantity L20001 Luna-FL™ Automated Fluorescence Cell Counter L12001 Cell Counting Slides, 50 Slides (100 Counts) 1 box L12002 Cell Counting Slides, 500 Slides (1,000 Counts)
  • Page 80 For more information or technical support, please call, write, fax, or email. Our regional suppliers are listed on our web page (www.logosbio.com). Logos Biosystems, Inc. Address: STE 930 Doosan Venturedigm, 126-1 Pyungchon-Dong, Dongan-Gu, Anyang-City, Gyunggi-Do, Republic of Korea (Zip code: 431-755)

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