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Logos LUNA-FL User Manual

Automated fluorescence cell counter
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LUNA-FL™ Automated Fluorescence Cell Counter
User Manual
Catalog #: L20001
Document #: LBSM-MD-ML-LUF-001
Version: VL1712-01
1
www.logosbio.com

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Summary of Contents for Logos LUNA-FL

  • Page 1 LUNA-FL™ Automated Fluorescence Cell Counter User Manual Catalog #: L20001 Document #: LBSM-MD-ML-LUF-001 Version: VL1712-01 www.logosbio.com...

  • Page 2: Table Of Contents

    IMPORTANT LUNA-FL™ Automated Fluorescence 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 8.5 Managing Protocols ---------------------------------------------------------------------------------- Chapter 9 – Maintenance and Troubleshooting ----------------------------------- 9.1 Cleaning ------------------------------------------------------------------------------------------------- 9.2 Changing the Battery --------------------------------------------------------------------------------- 9.3 Calibrating the Counter ------------------------------------------------------------------------------ 9.4 Updating Firmware ----------------------------------------------------------------------------------- 9.5 Troubleshooting --------------------------------------------------------------------------------------- Chapter 10 – Ordering Information --------------------------------------------------------- Chapter 11 – Purchaser Notification -------------------------------------------------------- www.logosbio.com...

  • Page 5: Safety Information

    Safety Information For the best results, users of the LUNA-FL™ Automated Fluorescence 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 6 <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 7: General Guidelines

    General Guidelines for Using the LUNA-FL™ Automated Fluorescence Cell Counter In order to achieve the best results with the LUNA-FL™ Automated Fluorescence 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 8: Environment Conditions

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

  • Page 9: 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 10 Number & percentage of the GFP-transfected cells The PhotonSlide™ 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.
  • Page 11 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 12: Product Contents

    USB Drive, 16 GB 1 unit Note: The PhotonSlide™ is a counting slide developed by Logos Biosystems that provides much better fluorescence signal, with ultra-low autofluorescencence. Users of the LUNA-FL™ are recommended to use the PhotonSlide™, especially for the yeast counting and GFP transfection assay.

  • Page 13: Product Specifications

    1.3 Product Specifications 1.3.1. LUNA-FL™ cell counter specifications Instrument Type Bench-top cell counter Dimensions (WxDxH) 22 x 21 x 9 cm Weight 1.8 kg (without the power cord/adapter) Light Source Excitation Wave Length 470 +/- 20 nm (Blue) Emission Wave Length...

  • Page 14: Product Description

    1.4.2 Rear view of the LUNA-FL™ Automated Fluorescence Cell Counter The rear view of the LUNA-FL™ Automated Fluorescence 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 15 1.4.3 Right side view of the LUNA-FL™ Automated Fluorescence Cell Counter The right side view of the LUNA-FL™ Automated Fluorescence 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 16 1.4.5 The PhotonSlide™ for the LUNA-FL™ The PhotonSlide™ consists of two chambers, labeled as A and B, that can be used for the same sample as duplicates or for two different samples (for example, the GFP transfection assay). The depth of the counting chamber is 100 µm.

  • Page 17: 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 Fluorescence 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 18: 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 4 options as shown below. Users can choose one of them to perform. <Start-Up Screen of the LUNA-FL™ Automated Fluorescence Cell Counter> 2.2.2 Bright Field Cell Counting Screen...
  • Page 19 2.2.3 Fluorescence Cell Counting Screen 2.2.3.1 Fluorescence Counting – Bright Field Screen 2.2.3.2 Fluorescence Counting – Green Fluorescence Screen 2.2.3.3 Fluorescence Counting – Red Fluorescence Screen www.logosbio.com...
  • Page 20 2.2.4 GFP Transfection Assay 2.2.4.1 GFP Transfection Assay – Bright Field Screen 2.2.4.2 GFP Transfection Assay – Green Fluorescence Screen www.logosbio.com...
  • Page 21 2.2.5 Yeast Cell Counting Screen 2.2.5.1 Yeast Cell Counting – Bright Field Screen 2.2.5.2 Yeast Cell Counting – Green Fluorescence Screen 2.2.5.3 Yeast Cell Counting – Red Fluorescence Screen www.logosbio.com...

  • Page 22: 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 4 counting screen options.

  • Page 23: 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 24 “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-”. www.logosbio.com...

  • Page 25: Bright Field Counting Options

    “1”. Note: The cell counting parameters of the “Bright Field Cell Counting” mode of the LUNA-FL™ is optimized with the use of Trypan Blue. Although the counting “Without Trypan Blue” option is provided in the protocol menu to meet mostly the “total cell counting”...
  • Page 26 results. <Dilution Factor Check> Note: The setting of the “Dilution Factor” must be checked and done manually. Custom staining In this option, cell samples can be stained with Erythrosin B Stain or other dyes (or even the culture media) for counting. Before choosing this option, the instrument must be re-calibrated with users’...
  • Page 27 2.6.3 “Auto Exposure” function of LUNA-FL™ can be used on the software version 3.0 or higher. With only “With Trypan Blue (1:1)” options, auto exposure function can be applied. As a default, “Auto Exposure” button is activated (On). The description of “Auto Exposure” option is as below...

  • Page 28: 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 Sub-option Description The default value is “off.” When it is turned on, the counting result Result Display box displays only the total cell number, not showing live/dead cells. The default value is “Total Cells (on).”...

  • Page 29: Chapter 3 - Bright Field Counting Cells

    3.1.3 Mix 10 µl of the cell sample with 10 µl 0.4% 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 the LUNA-FL™ is optimized with the use of Trypan Blue Stain. Although the counting “Without Trypan Blue” option is provided in the protocol menu (section 2.6.2) to meet mostly the “total cell counting”...

  • Page 30: Counting Cells

    The instrument analyzes only the first inserted chamber. Gently insert the counting slide to the end. Note: After inserting the slide, the 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 31 <Preview Screen> Note: Users must find an accurate focus to get the best cell counting results. Note: To get the best focus, the image can be magnified by using the “Zoom-in” button located under the “Count” button. Initially, the “Zoom-in” button is set to a 1x image. When pressed once, a 2x image appears.
  • Page 32 <Wrong Staining Option Selected: Too Dark Preview Image> Note: The default preview image is set to a grey scale. To change the preview image to a color mode, press the “Color” button once, and the preview image will turn to a color image and the “Color button” will change to bright blue.
  • Page 33 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 34: 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 Fluorescence Cell Counter because it allows the user to review the data to determine the accuracy of the counting on-site without depending on a computer.

  • Page 35: 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.
  • Page 36 <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 37 <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 38 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 39: Using The Dilution Calculator

    3.6 Using the Dilution Calculator 3.6.1 The LUNA-FL™ Automated Fluorescence 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 40 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. www.logosbio.com...

  • Page 41: 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 42 USB drive. 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 43: Printing The Counting Results

    <LUNA™ Printer> USB Connection Port Note: The LUNA™ Printer and Paper are specially designed for the LUNA-FL™ and must be purchased only from Logos Biosystems or its local distributors. Note: For more information on the LUNA™ Printer, please see the User Manual of the LUNA™ Printer.
  • Page 44 <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. www.logosbio.com...
  • Page 45 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> www.logosbio.com...

  • Page 46: 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  PhotonSlide™ (Cat # L12005) or LUNA™ Reusable Slide (Cat # L12008)  Acridine Orange/Propidium Iodide Stain (Cat # F23001) 4.1.2 If desired, insert a USB drive to save data and results.

  • Page 47: Counting Cells

    <Slide Inserting> 4.2.2 The remaining 10 µl of the sample can be used for another duplicate counting to get 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 10 sec-1 min (depending on the sample condition) in order for the cells to settle down. This step is very important.
  • Page 48 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 49 Please note that the touch screen of the LUNA-FL™ Automated Fluorescence Cell Counter is a resistive touch screen, which needs a bit of pressure in order to get a response.
  • Page 50 Note: To get the best focus, the image can be magnified by using the “Zoom-in” button located 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.
  • Page 51 4.3.8 Within approximately 30 seconds (depending on cell concentration), the image of the cell sample and data/results (Total, Live, Dead, Viability, avg. size, total number, live number and dead number) will be displayed on the screen. Note: The LUNA-FL™ take 3 images from the bright field, green, and red channel sequentially. www.logosbio.com...
  • Page 52 <Fluorescence Cell Counting Results> www.logosbio.com...

  • Page 53: 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 www.logosbio.com...
  • Page 54 Note: This “Tag” function is one of the distinct tools of the LUNA-FL™ Automated Fluorescence 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 55: 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.
  • Page 56 <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 57 <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 58 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 59: Using The Dilution Calculator

    4.6 Using the Dilution Calculator 4.6.1 The LUNA-FL™ Automated Fluorescence 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 60 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. www.logosbio.com...

  • Page 61: 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 62 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 63: Printing The Counting Results

    4.8 Printing the Counting Results Please see Section 3.8 to print the counting results. www.logosbio.com...

  • Page 64: Chapter 5 - Gfp Transfection Assay

    PhotonSlide™ (Cat No. L12005) or LUNA™ Reusable Slide (Cat # L12008)  A USB drive to save data and results, if desired. Note: The reagents and kits for the GFP transfection assay are not included in the LUNA-FL™ package. They must be purchased separately from other suppliers. www.logosbio.com...

  • Page 65: Loading Samples Into Slides

    5.2 Loading Samples into Slides 5.2.1 Holding the edge of the slide, load 10 µl of the negative control on one side and the GFP- transfected sample on the other side of the counting slide. For easy and accurate loading, tilt the pipette around 45-60 degrees as shown below.

  • Page 66: Measuring Gfp Transfection Efficiency

    The instrument analyzes only the first inserted chamber upon counting. 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 67 The cell image can be navigated by touching the screen and moving a finger or stylus pen on the image. Please note that the touch screen of the LUNA-FL™ Automated Fluorescence Cell Counter is a resistive touch screen type, which needs a bit of pressure in order to get a response.
  • Page 68 <Adjusting the Focus> Note: To get the best focus, the image can be magnified by using the “Zoom-in” button located under the “Count Sample” 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 is shown.
  • Page 69 <Green Fluorescence Preview in Measuring GFP Transfection Efficiency > Note: The exposure level of the green fluorescence must be started from 18 and then, depending on the signal strength, it can be adjusted. 5.3.2 Setting the negative control 5.3.2.1 Remove the slide containing the transfected sample and insert a slide containing the negative control.
  • Page 70 5.3.2.3 Within approximately 30 seconds (depending on cell concentration), the analysis of the negative control will be done. Press the “OK” button. 5.3.3 Counting the GFP transfected sample 5.3.3.1 Insert a slide containing the GFP-transfected sample again and press the “Count Sample” button.
  • Page 71 <Result Screen> 5.3.3.3 If you have multiple GFP transfected samples and want to measure the efficiency of the next sample, insert the new slide and press the “Next Count” button. 5.3.3.4 Press the “Count Sample” button as needed. www.logosbio.com...

  • Page 72: Using The "Tag" Function

    “Images” and “Tag” buttons as shown below. <Result Screen> Note: This “Tag” function is one of the distinct tools of the LUNA-FL™ Automated Fluorescence Cell Counter because it helps users review and determine the accuracy of measuring GFP transfection efficiency on-site without depending on a separate computer.
  • Page 73 <Tagged Cell Image> Press Tag button Green circle – GFP transfected cell 5.4.3 After reviewing the accuracy of the image analysis, “Tag” button can be pressed again to remove the green circles. www.logosbio.com...

  • Page 74: Gfp Intensity And Number Distribution

    <Graphical Representation of the Distribution of GFP Intensity > Functional buttons in the menu “Count  Graph/Gating” 1) “Control + Sample” 2) “Control” button 3) “Sample” <GFP Intensity Threshold> GFP Intensity Threshold is automatically determined by the LUNA-FL™ software. www.logosbio.com...
  • Page 75 <Percentages of GFP (-) and GFP (+) cells> Percentages of GFP (-) and GFP (+) cells of the sample are shown here. Note: The default threshold automatically set by the instrument is the smallest GFP intensity value, where 100% of negative control cells are classified as GFP (-) cells. In case the threshold drawn automatically is not satisfactory, users can re-assign the threshold as shown in the next figure to meet their specific needs.
  • Page 76 <User-defined GFP Intensity Threshold 2> GFP Intensity Threshold can also be increased by users. Users can touch the right arrowhead icon located in the lower left corner. To update the percentage values of GFP (-) and GFP (+) cells, the “Apply”...

  • Page 77: On/Off Of The Bright And Green Channel

    5.6 On/off of the Bright and Green Channel 5.6.1 Users can turn on or off of the bright and green channels each by clicking the “On/Off” button. <Bright Field (on) and Green (on)> <Bright Field (off) and Green (on)> www.logosbio.com...

  • Page 78: Saving The Image And Generating A Report Of The Current Count

    5.6.2 Users can adjust the level of the bright and green channels each by clicking the lowering arrowhead icon. <The level of Bright Field was lowered.> 5.7 Saving the Image and Generating a Report of the Current Count 5.7.1 To save data and/or generate a report, insert a USB drive into the USB port on the left side of the instrument.
  • Page 79 <Save and Report Screen> Note: This “Save” window contains three check boxes as follows:  Analyzed Image: Check this box to save an analyzed image. Live cells are tagged in green and dead cells in red for further analysis.  Raw Image: This box must always be checked in order to save the image in TIF format.

  • Page 80: Printing The Counting Results

    <Images and Report Saving> Note: With the LUNA-FL™ software, additional graphical representations of the data and results are provided in the PDF report, as shown below. <Two page PDF report generated by LUNA-FL> page – Fluorescence Intensity Histograms> <1 page - Data and Results>...

  • Page 81: Chapter 6 - Yeast Cell Counting

    Chapter 6 – Yeast Cell Counting With a new reagent, Fluorescein Diacetate (FDA) and the software version 2.0 or higher, users of the LUNA-FL™ Automated Fluorescence Cell Counter can count yeast cells. For more information, please refer to the application note on the website (www.logosbio.com).
  • Page 82 is a good starting point. If needed, centrifuge the sample, remove the supernatant and resuspend the pellet with the yeast dilution buffer. After the dilution or washing step, proceed to the next step. IMPORTANT If your yeast cells were grown in the YPD medium, be sure to dilute the sample at least 1:100 with the Cell Dilution Buffer before counting.

  • Page 83: Loading Samples Into Slides

    6.2 Loading Samples into Slides 6.2.1 Holding the edge of the slide, load 10 µl of the mixed cell sample into the inlet of one chamber of the counting slide. For easy and accurate loading, tilt the pipette around 45-60 degrees as shown below. <Sample Loading>...

  • Page 84: Counting Yeasts

    <Slide Inserting> Note: After inserting the slide, the LUNA-FL™ only reads the first chamber. To read the second chamber, it must be taken out, rotated, and inserted again. Note: Make sure that the counting slide is not inserted upside-down. This may lead to sample spilling and could damage the counter.
  • Page 85 6.3.3 In the preview screen of the bright field mode, watch the yeast cells and wait for 30 sec-1 min (depending on the sample condition) in order for the cells to settle down. This step is very important because the yeast cells are very small and take some time to make a good focal plane. 2X or 4X zoom- in is helpful to focus.
  • Page 86 Note: Users can set the exposure level between 18 and 20 and the threshold between 1 and 5. Make sure that you start with 18 (exposure) and 2 (threshold) and then empirically determine the parameters. 6.3.5 Touch the “Count” button. <Yeast Counting Results>...
  • Page 87 Live cell concentration: 7.70 x 10e6 cells/mL Dilution factor: 1.11 Dead cell concentration: 0.00 x 10e0 cells/mL Min. cell size: 3 µm Viability: 100.0 % Max. cell size: 60 µm Average cell size: 9.0 µm Size gating: 3 - 60 µm Total cell number: 3351 Green fluorescence threshold: 5 Live cell number: 3351...
  • Page 88 <Adjusting the “Max” size> Note: By clicking the “Total/Live/Dead” button, users can see only one of the total, live, or dead cell groups as below. <Live Cells Only> www.logosbio.com...
  • Page 89 <Dead Cells Only> Note: By clicking the “Cluster Map” button, users can see the grouping status of the “Cluster Map” as below. <Cluster Map> www.logosbio.com...

  • Page 90: Using The "Tag" Function

    6.4.1 To verify the results after performing the counting results, press the “Images” and “Tag” buttons as shown below. <Result Screen> Note: This “Tag” function is one of the distinct tools of the LUNA-FL™ Automated Fluorescence Cell Counter because it helps users review and determine the accuracy on-site without depending on a computer.
  • Page 91 <Tagged Cell Image> Press Tag button Green circle – Live yeasts Red circle – Dead yeasts Note: In the scree above, the exposure level of each light channel can be changed by adjusting the arrowhead buttons. <Turn-off of the Bright Field Channel> 6.4.3 After reviewing the accuracy of the image analysis, “Tag”...

  • Page 92: Trouble Shooting

    → The Fluorescence Signal Enhancer 1 (Cat # F23213) can be used. See the following protocol. <Using the Fluorescence Signal Enhancer 1 (Cat # F23213)> 1. Dilute yeast cells up to 1/100 with the LUNA-FL™ dilution buffer. Or, centrifuge the yeast culture and resuspend in the LUNA-FL™ dilution buffer.

  • Page 93: Printing The Counting Results

    6.6 Printing the Counting Results Please see Section 3.8 to print the counting results. www.logosbio.com...

  • Page 94: Chapter 7 - Reviewing Previous Results

    Chapter 7 – Reviewing Previous Results The LUNA-FL™ Automated Fluorescence Cell Counter provides an easy-to-use review function using the previous count results saved in the USB drive for further review. This “Review” function applies to all 4 counting categories. 7.1 Importing Previous Counts 7.1.1 Insert the USB drive containing the previous results into the USB port of the instrument.

  • Page 95: Reviewing Previous Results

    7.2 Reviewing Previous Results 7.2.1 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> 7.2.2 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 96: Previous Count" Button

    7.3.1 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 97 <”Previous Count” Window for GFP Transfection Assay> 7.3.2 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. First insert a USB drive and then click the “Export to USB (.CSV)”...

  • Page 98: Chapter 8 - Setting Up The Protocol For Counting

    Chapter 8 – 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 99 The adjustable parameters are described as follows: 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 “2 x A”.
  • Page 100 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. The unit is 1 micrometer. www.logosbio.com...

  • Page 101: Adjusting Parameters For Fluorescence Counting

    8.2 Adjusting Parameters for Fluorescence Counting <Fluorescence Counting Parameters on the Protocol Menu> The adjustable parameters are described as follows: Parameter Range Default Description The default value for the dilution factor is “1.11”. In the protocol of the “Fluorescence Cell Counting” mode, 2 µl of the Acridine Orange/Propidium Iodide Stain (cat # F23001) is added to 18 µl of the cell sample.
  • Page 102 Maximum Use this parameter to set the maximum cell size for 1-90 Cell Size inclusion. The unit is 1 micrometer. Green Green and Red Fluorescence Threshold will determine Fluorescence 1-10 the level of threshold during the image processing. Threshold Increased threshold will detect less cells by subtracting the background more stringently.

  • Page 103: Adjusting Parameters For Yeast Cell Counting

    8.3 Adjusting Parameters for Yeast Cell Counting <Yeast Counting Parameters on the Protocol Menu> The adjustable parameters are described as follows: Parameter Range Default Description The default value for the dilution factor is “1.11”. To perform the yeast counting, 1 µl of FDA and 1 µl of PI is added to 18 µl of the yeast sample.
  • Page 104 the background more stringently. Decreased threshold can detect more cells, but will increase the number of noise signal. Fluorescence 1-10 Threshold For yeast, it is recommended to start with 2 or 3, depending on the signal strength. www.logosbio.com...

  • Page 105: Adjusting Parameters For Gfp Transfection Assay

    8.4 Adjusting Parameters for GFP Transfection Assay <Parameters for GFP Transfection Assay on the Protocol Menu> The adjustable parameters are described as follows: Parameter Range Default Description The default value for the dilution factor is “1”. Users can modify this value according to the dilution of the original samples.

  • Page 106: Managing Protocols

    Reduction counting. Higher noise reduction will not detect faint signals and weakly stained objects. Lower noise reduction means increasing the sensitivity of the objects and detecting faint signals. Adjust the noise reduction to the appropriate level. It is also useful when cells are over- stained or under-stained with Trypan Blue stain.
  • Page 107 After saving, users can load the protocol for future counting. Note: The LUNA-FL™ Automated Fluorescence Cell Counter is provided with a factory setting protocol named “DEFAULT”. This “DEFAULT” protocol cannot be edited or deleted. The parameters in the “DEFAULT” protocol have been tested with many different cell types and can be used for a wide variety of cells without changing parameters.

  • Page 108: Chapter 9 - 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 109: Changing The Battery

    9.2 Changing the Battery The battery of the LUNA-FL™ Automated Fluorescence 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 the service personnel of the instrument supplier in your country.

  • Page 110: Calibrating The Counter

    9.3 Calibrating the Counter In general, recalibration of the LUNA-FL™ Automated Fluorescence 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. 9.3.1 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 111 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. 9.3.4 “Calibration Step 2” is necessary for the “Custom Staining Calibration.” From this custom calibration, users can use their own stains for cell counting.
  • Page 112 0.1% Trypan Blue concentration, users can mix cells with 0.2% Trypan Blue Stain 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. www.logosbio.com...
  • Page 113 <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. 9.3.6 When the three calibration steps are done successfully, users will see the message box as below.
  • Page 114 <Successful Calibration> www.logosbio.com...

  • Page 115: Updating Firmware

    <Settings> 9.4.2 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 116: Troubleshooting

    AO and PI. Too low or too high Make sure fluorescence settings in the preview fluorescence setting screen are in a reasonable range. Inaccurate calibration Every LUNA-FL™ instrument has been pre- www.logosbio.com...
  • Page 117 The more cells that are counted, the more precise the result. Low precision range Although LUNA-FL™ can count as low as 5 x 10 cells/mL, cell concentration higher than 2 x 10 cells/mL is required for 10% or lower Coefficient for variation (CV).
  • Page 118 correctly. Last, try the update again. If the problem continues, contact your local supplier. www.logosbio.com...

  • Page 119: Chapter 10 - Ordering Information

    Chapter 10 – Ordering Information The following products can be ordered from your regional supplier or the website (www.logosbio.com). Cat # Product Quantity LUNA-FL™ Automated Fluorescence Cell Counter Counter L20001 1 unit PhotonSlide™, 50 Slides L12005 1 box PhotonSlide™, 500 Slides...

  • Page 120: Chapter 11 - Purchaser Notification

    Logos Biosystems, Inc. (“Company”) will not claim any consideration against the purchaser of infringement of patents owned or controlled by the Company which cover the product based on the manufacture, use or sale of a...
  • Page 121 used or contaminated in some labs, including but not limited to, HIV or other infectious disease or blood-handling labs. This limited warranty does not cover refund, replacement, and repair incurred by accident, abuse, misuse, neglect, unauthorized repair, or modification of the Instrument. This limited warranty will be invalid if the Instrument is disassembled or repaired by the Purchaser.
  • Page 122 Logos Biosystems HEADQUARTERS FL 2 & 3 28 Simindaero 327beon-gil, Dongan-gu Anyang-si, Gyeonggi-do 14055 OUTH OREA Tel: +82 31 478 4185 Fax: +82 31 478 4184 Email: sales@logosbio.com 7700 Little River Turnpike STE 207 Annandale, VA 22003 Tel: +1 703 622 4660...

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