logos biosystems Luna-FL User manual

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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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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 -------------------------------------------------------------------------------------------- 4
General Guidelines ------------------------------------------------------------------------------------------- 6
Environment Conditions ------------------------------------------------------------------------------------- 7
Chapter 1 – Introduction --------------------------------------------------------------------------- 8
1.1 Product Overview ------------------------------------------------------------------------------------- 8
1.2 Product Contents -------------------------------------------------------------------------------------- 11
1.3 Product Specifications ------------------------------------------------------------------------------- 12
1.4 Product Description ----------------------------------------------------------------------------------- 13
Chapter 2 – Setting up ------------------------------------------------------------------------------- 16
2.1 Installation ---------------------------------------------------------------------------------------------- 16
2.2 Start-Up Screen --------------------------------------------------------------------------------------- 17
2.3 Settings -------------------------------------------------------------------------------------------------- 19
2.4 Setting the Date and Time -------------------------------------------------------------------------- 20
2.5 Save Options ------------------------------------------------------------------------------------------ 20
2.6 Counting Options ------------------------------------------------------------------------------------- 21
2.7 Result/Printer/Calculator Options ----------------------------------------------------------------- 23
Chapter 3 – Bright Field Counting Cells -------------------------------------------------- 24
3.1 Sample Preparation ---------------------------------------------------------------------------------- 24
3.2 Loading Samples into Slides ----------------------------------------------------------------------- 24
3.3 Counting Cells ----------------------------------------------------------------------------------------- 25
3.4 Using the “Tag” Function ---------------------------------------------------------------------------- 29
3.5 Cell Size and Number Distribution ---------------------------------------------------------------- 30
3.6 Using the Dilution Calculator ----------------------------------------------------------------------- 34
3.7 Saving Images and Generating a PDF Report of the Current Count--------------------- 36
3.8 Printing the Counting Results ---------------------------------------------------------------------- 38

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Chapter 4 – Fluorescence Cell Counting ------------------------------------------------- 41
4.1 Sample Preparation ---------------------------------------------------------------------------------- 41
4.2 Loading Samples into Slides ----------------------------------------------------------------------- 41
4.3 Counting Cells ----------------------------------------------------------------------------------------- 42
4.4 Using the “Tag” Function ---------------------------------------------------------------------------- 48
4.5 Cell Size and Number Distribution --------------------------------------------------------------- 50
4.6 Using the Dilution Calculator ----------------------------------------------------------------------- 54
4.7 Saving Images and Generating a PDF Report of the Current Count --------------------
4.8 Printing the Counting Results ----------------------------------------------------------------------
Chapter 5 – Reviewing Saved Count Results -----------------------------------------
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5.1 Importing Previous Counts ------------------------------------------------------------------------- 59
5.2 Reviewing Previous results ------------------------------------------------------------------------ 60
5.3 “Previous Count” button ----------------------------------------------------------------------------- 61
Chapter 6 – Setting Up the Protocol for Counting --------------------------------- 63
6.1 Adjusting Parameters for Bright Field Counting ---------------------------------------------- 63
6.2 Adjusting Parameters for Fluorescence Counting --------------------------------------------
6.3 Managing Protocols ----------------------------------------------------------------------------------
Chapter 7 – Maintenance and Troubleshooting -----------------------------------
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7.1 Cleaning ------------------------------------------------------------------------------------------------- 67
7.2 Changing the Battery --------------------------------------------------------------------------------- 68
7.3 Calibrating the Counter ------------------------------------------------------------------------------ 69
7.4 Updating Firmware -----------------------------------------------------------------------------------
7.5 Troubleshooting ---------------------------------------------------------------------------------------
Chapter 8 – Ordering Information ------------------------------------------------------------
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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. Do not place it in a humid environment such as an incubator.
For operating environment, see page 7.
2. Trypan blue stain, acridine orange, propidium iodide, and other reagents are known as
hazardous materials. While handling the solution, always wear gloves to avoid exposure.
3. Before use, make sure that the input voltage is compatible with the instrument’s power supply
voltage.
4. For optimal operations, place the instrument on a flat bench and avoid any vibration.
5. Turn on the instrument only after connecting both ends of its power cord to the wall outlet as
well as the instrument. Always turn off the instrument before disconnecting the power cord
and/or moving the instrument.
6. Ensure that the power cord is firmly plugged into the power inlet, the wall outlet and AC adapter.
7. When the instrument is operating for a long time, its temperature can become too high. Please
be careful that the instrument’s temperature does not become too high during long and
continuous operation times. When operating, leave enough space around the instrument so
there is enough room for air circulation and cooling.
8. Do not disassemble the instrument in any event. If the instrument is out of order or dropped or
broken, please contact a service person. Disassembling the instrument invalidates its
warranty.
9. Use only authorized components (adaptor, power cord, and USB drive).
10. If the instrument emits smoke, disconnect the power cord immediately from the wall outlet and
contact a service person.
11. Used counting slides must be disposed as biohazard waste.

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<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.
The CE mark indicates that this instrument conforms to all applicable European
Community provisions for which this marking is required. Users must be aware of
and follow the conditions described in this manual for operating the instrument.
The protection provided by the instrument may be impaired if the instrument is
used in a manner not specified by this manual.
Protective earth (Ground)

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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.
1. The instrument must be operated in compliance with the environmental conditions described on
page 7. In particular, the temperature and humidity conditions are important.
2. Samples must be handled in an appropriate way, depending on user’s requirements.
3. Hold the Luna-FL™ cell counting chamber slide by the edges to avoid touching its optical
surface. Make sure that no damage or contamination occurs on the optical surfaces of the slide.
4. After mixing the cell sample with the supplied reagents, perform cell counting within 1-3
minutes for accurate cell viability measurements. Optimally, count your sample at least twice
(duplicate readings) to obtain better results.
5. Since the Luna-FL™ is calibrated before shipping, users generally do not need to re-calibrate
before use. However, if re-calibration is needed, for example due to long-term use, please refer
to Section 7.3 Calibrating the Counter.
6. Do not touch trypan blue solution and other reagents with bare hands, as they are hazardous
chemicals. After using chamber slides, dispose of them as hazardous waste. Do not reuse the
slides.

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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 2

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Chapter 1 – Introduction
1.1 Product Overview
The Luna-FL™ automated cell counter is a small, fast, and affordable image-based cell counting
device, containing bright field and dual fluorescence optics, that automatically counts various kinds
of cells for research use.
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™ provides a state of the art cell counting device and
eliminates the tedium and subjectivity of manual cell counting.
The Luna-FL™ can be used in a very simple procedure. For example, in order to accomplish dual
fluorescence counting, first, mix 18 l of the cell sample with 2 l of the AO/PI (Acridine Orange and
Propidium Iodide) from the cell viability kit (in case that there is not enough cell sample, 9 l of the
cell sample with 1 l of the AO/PI solution can be used to load the total 10 l in the counting
chamber). Second, load 10-12 l of the mixed cell suspension into the Luna-FL™ cell counting slide.
Third, insert the slide into the slide port of the instrument and adjust the focus knob to get an
appropriate cell image. Last, press the “Count” button and the results of cell count and viability will
be displayed on the screen. The counting image can be downloaded onto a USB drive in a TIF
format for future analysis.
<AO/PI Stained Cell Image>
Assay principle: AO stains all cells, and PI stains dead cells only. If AO and PI exist together, AO
fluorescence is mostly quenched by PI. Therefore, AO fluorescence (green) is found in live cells
only, and PI fluorescence (red) is found in dead cells only. Cell viability analysis using AO/PI double
staining is a proven and accepted method.

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The Luna-FL™ automated cell counter provides the data below.
 Number of live and dead cells and their concentrations
 Number of total cells and their concentrations
 Viability percentage (% live cells to total cells)
 Cell images (showing live cells as green circles and dead cells as red circles)
 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.
Key features of the Luna-FL™ are as follows:
Key features Description
Bright field & dual fluorescence
optics
The Luna-FL™ integrates bright field, as well as dual
fluorescence optical components to provide advanced cell
counting functionalities. The cell image acquired from each
channel (bright, green, and red) can be merged directly on the
screen. The brightness of each color can be adjusted
independently for accurate monitoring.
Small footprint
Due to its minimal size (22 cm x 21 x 9 cm) & weight (1.8 kg),
it can be used on a clean bench or stored in a biosafety
cabinet for convenience.
Accuracy & precision
Due to its sophisticated optical components and counting
algorithm, Luna-FL™ cell counter provides reproducible
results every time.
Easy-to-operate user interface The intuitive user interface based on a touch screen enables
simple and easy operation.
Shortest time-to-results Results for most cell lines are available within 10-30 seconds
of pressing the ‘Count’ button.
Innovative counting slide
Luna-FL™ cell counter adopts an innovative counting slide
made with “T-bond” technology without using hazardous
organic solvents.

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Cell concentration & viability range
Measurements can be made for cells at concentrations within
the 1x104 to 1x107 (preferably 5x104 to 1x107) cells/ml range
and for cells within the 1-90m (preferably, 5-60m) diameter
range.
Cell size gating After counting, users can set the gating parameter for cell
size.
Easy-to-use dilution calculator Dilution concentration can easily be calculated using the built-
in calculator.
Set up & maintenance Just plug in and it is ready for use, with virtually no
maintenance time or costs.
Counting image transfer The counting image can be downloaded onto a USB drive in
TIF (Tag Image File) format for later use or review.
Individual protocol saving Each user can set or adjust counting parameters and save the
counting protocol for later use.
Automatic reporting
Luna-FL™ cell counter provides a counting report based on
the count results in PDF (Portable Document Format)
immediately after performing a cell count.

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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
Trypan blue stain (0.4 %) 2 x 1 ml
Luna-FL™ calibration beads 0.5 X 1 ml
Luna™ USB drive (4GB) 1 unit
When you receive the product package, please check that all the components listed above are
included and that no damage has occurred during transit. The warranty does not cover damage that
may occur during the shipping and handling process. Any damage claims must be filed with the
carrier.

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1.3 Product Specifications
1.3.1. Luna-FL™ cell counter specifications
Instrument Type Bench-top cell counter
Dimensions (WxDxH) 22 cm x 21 cm X 9 cm
Weight 1.8 kg (without the power cord/adapter)
Light Source LED
Excitation Wave Length 470 +/- 20 nm (Blue)
Emission Wave Length 525 +/- 25 nm (Green), 600 LP (Red)
Cell Concentration Range 1x104 ~ 1x107 (optimally 5x104 ~ 1x107) cells/ml
Cell Diameter Range 1~90 (optimally 5~60) m
Cell Circularity Range 30~100%
Cell Viability Range 0~100%
Image Resolution CMOS camera (5 MP)
Image Type TIF
Reporting PDF report, .CSV file
LCD Display 7 inches (800 x 480 pixels)
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
Chamber Volume 10 l
1.3.3 Luna-FL™ software
Cell Counting Bright field cell counting
Dual fluorescence cell counting

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1.4 Product Description
1.4.1 Front view of the Luna-FL™ automated cell counter
The front view of the Luna-FL™ automated cell counter shows a wide touch screen. This interface
contains buttons for all functions and displays results.
<Front View>
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. Be sure to check the electrical outlet configuration in your
country.
<Rear View>

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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. The slide port is used to insert the counting chamber slide
loaded with the sample into the instrument.
<Right Side View>
1.4.4 Left side view of the Luna™ automated cell counter
On the left side of the cell counter, there is a USB drive port into which a USB drive is inserted for
transferring and saving data. One USB drive is supplied in the product package. Any standard USB
drive can be used.
<Left Side View>
Focus knob
Counting slide port
USB drive port

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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. The counted cell volume is about 0.5 l, almost the same as counting
five (1 mm x 1 mm) squares in a standard hemocytometer.
Note: 0.4% trypan blue solution is blue and the AO/PI solution is pink (or orange).
Sample loading chamber (marked as A and B)
Sample loading port

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Chapter 2 – Setting up
2.1 Installation
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.
2.1.4 Turn on the instrument using the power button located on the back of the cell counter.
2.1.5 The Start-Up screen will display on the touch screen as shown below in 2.2.

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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.
<Start-Up Screen of the Luna-FL™ Automated Cell Counter>
2.2.2 Bright Field Counting Screen

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2.2.3 Fluorescence Counting Screen
2.2.3.1 Green Fluorescence Counting Screen
2.2.3.2 Red Fluorescence Counting Screen

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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.
2.3.2 Press the “Settings” button ( ) located on the upper right corner of the counting screen. Then,
the following “Settings” screen appears. From here, users can change or adjust various preferences
or options.
<Settings>
2.3.3 The “Settings” menu allows you to perform the following:
 Background calibration of the instrument (described in Section 7.3 Calibrating the Counter)
 Firmware update for the installation of new versions of the firmware that will be released
from time to time (described in Section 7.4 Updating the Firmware)
 Date and time set up
 Other options such as “Save”, “Counting”, and “Result/Printer/Calculator”

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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. .
2.5 Save Options
2.5.1 The “Save Options” screen will be seen as below, after selecting the “Save Options” button.
As a default, only the “Analyzed Image” button is activated (On), while the “Raw Image” and “Report”
button are inactivated (Off).
<Save Options>
2.5.2 In the “Default Image Save” menu, users can choose their options to save the raw image,
PDF report, or both. Each button can be switched on and off.
Saved Items Description
Analyzed image The image contains the counting results and the tagging of live and
dead cells.
Raw image The image contains only the captured image for counting. In the
bright field cell counting mode, the bright field image will be