Logos LUNA-FL User manual

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

2
www.logosbio.com
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 --------------------------------------------------------------------------------------------
5
General Guidelines -------------------------------------------------------------------------------------------
7
Environment Conditions -------------------------------------------------------------------------------------
8
Chapter 1 –Introduction ---------------------------------------------------------------------------
9
1.1 Product Overview -------------------------------------------------------------------------------------
9
1.2 Product Contents --------------------------------------------------------------------------------------
12
1.3 Product Specifications -------------------------------------------------------------------------------
13
1.4 Product Description -----------------------------------------------------------------------------------
14
Chapter 2 –Setting up -------------------------------------------------------------------------------
17
2.1 Installation ----------------------------------------------------------------------------------------------
17
2.2 Start-Up Screen ---------------------------------------------------------------------------------------
18
2.3 Settings --------------------------------------------------------------------------------------------------
22
2.4 Setting the Date and Time --------------------------------------------------------------------------
23
2.5 Save Options ------------------------------------------------------------------------------------------
23
2.6 Bright Field Counting Options ---------------------------------------------------------------------
25
2.7 Result/Printer/Calculator Options -----------------------------------------------------------------
28
Chapter 3 –Bright Field Counting Cells --------------------------------------------------
29
3.1 Sample Preparation ----------------------------------------------------------------------------------
29
3.2 Loading Samples into Slides -----------------------------------------------------------------------
29
3.3 Counting Cells -----------------------------------------------------------------------------------------
30
3.4 Using the “Tag” Function ----------------------------------------------------------------------------
34
3.5 Cell Size and Number Distribution ----------------------------------------------------------------
35
3.6 Using the Dilution Calculator -----------------------------------------------------------------------
39
3.7 Saving Images and Generating a PDF Report of the Current Count---------------------
41
3.8 Printing the Counting Results ----------------------------------------------------------------------
43

3
www.logosbio.com
Chapter 4 –Fluorescence Cell Counting -------------------------------------------------
46
4.1 Sample Preparation ----------------------------------------------------------------------------------
46
4.2 Loading Samples into Slides -----------------------------------------------------------------------
46
4.3 Counting Cells -----------------------------------------------------------------------------------------
47
4.4 Using the “Tag” Function ----------------------------------------------------------------------------
53
4.5 Cell Size and Number Distribution ---------------------------------------------------------------
55
4.6 Using the Dilution Calculator -----------------------------------------------------------------------
59
4.7 Saving Images and Generating a PDF Report of the Current Count --------------------
4.8 Printing the Counting Results ----------------------------------------------------------------------
Chapter 5 –GFP Transfection Assay -------------------------------------------------------
61
63
64
5.1 Sample Preparation ----------------------------------------------------------------------------------
64
5.2 Loading Samples into Slides -----------------------------------------------------------------------
65
5.3 Measuring GFP Transfection Efficiency ---------------------------------------------------------
66
5.4 Using the “Tag” Function ----------------------------------------------------------------------------
72
5.5 GFP Intensity and Number Distribution ---------------------------------------------------------
74
5.6 On/off of the Bright and Green Channel --------------------------------------------------------
77
5.7 Saving the Image and Generating a Report of the Current Count -----------------------
78
5.8 Printing the Counting Results ----------------------------------------------------------------------
80
Chapter 6 –Yeast Cell Counting --------------------------------------------------------------
81
6.1 Sample Preparation ----------------------------------------------------------------------------------
81
6.2 Loading Samples into Slides -----------------------------------------------------------------------
83
6.3 Counting Yeasts ---------------------------------------------------------------------------------------
84
6.4 Using the “Tag” Function ----------------------------------------------------------------------------
90
6.5 Trouble Shooting --------------------------------------------------------------------------------------
92
6.6 Printing the Counting Results ----------------------------------------------------------------------
93
Chapter 7 –Reviewing Previous Results ------------------------------------------------
94
7.1 Importing Previous Counts -------------------------------------------------------------------------
94
7.2 Reviewing Previous results ------------------------------------------------------------------------
95
7.3 “Previous Count” button -----------------------------------------------------------------------------
96
Chapter 8 –Setting Up the Protocol for Counting ---------------------------------
98
8.1 Adjusting Parameters for Bright Field Counting ----------------------------------------------
98
8.2 Adjusting Parameters for Fluorescence Counting --------------------------------------------
101
8.3 Adjusting Parameters for Yeast Cell Counting -------------------------------------------------
103
8.4 Adjusting Parameters for GFP Transfection Assay -------------------------------------------
105

4
www.logosbio.com
8.5 Managing Protocols ----------------------------------------------------------------------------------
Chapter 9 –Maintenance and Troubleshooting -----------------------------------
106
108
9.1 Cleaning -------------------------------------------------------------------------------------------------
108
9.2 Changing the Battery ---------------------------------------------------------------------------------
109
9.3 Calibrating the Counter ------------------------------------------------------------------------------
110
9.4 Updating Firmware -----------------------------------------------------------------------------------
9.5 Troubleshooting ---------------------------------------------------------------------------------------
Chapter 10 –Ordering Information ---------------------------------------------------------
115
116
119
Chapter 11 –Purchaser Notification --------------------------------------------------------
120

5
www.logosbio.com
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. Do not place it in a humid environment such as an incubator. For more
info, refer to Section Environmental Conditions.
2. Trypan Blue Stain, Acridine Orange Stain, Propidium Iodide Stain, 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.
12. The LUNA-FL™ Automated Cell Counter is an electrical laboratory instrument for scientific research
use only. It is not a medical, therapeutic, or in vitro diagnostics device.

6
www.logosbio.com
<Symbols used in this User Manual>
TheWEEE (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)

7
www.logosbio.com
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.
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 slides by the edges to avoid touching the 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 9.3 Calibrating the Counter.
6. Do not touch Trypan Blue Stain and other reagents with bare hands, as they are hazardous
chemicals. After using slides, dispose of them as hazardous waste. Do not reuse the slides.

8
www.logosbio.com
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

9
www.logosbio.com
Chapter 1 –Introduction
1.1 Product Overview
The LUNA-FL™ Automated Fluorescence 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 µl of the cell sample with 2 µl Acridine Orange/Propidium Iodide
Stain (in case that there is not enough cell sample, 9 µl of the cell sample with 1 µl of the PhotonSlide™
or LUNA™ Reusable Slide can be used to load the total 10 µl in the counting chamber). Second, load
10-12 µl of the mixed cell suspension into a PhotonSlide™ or LUNA™ Reusable 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.
<Acridine Orange/Propidium Iodide 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 Acridine
Orange/Propidium Iodide double staining is a proven and accepted method.

10
www.logosbio.com
The LUNA-FL™ Automated Fluorescence 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
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.
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 7-30 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.
Cell concentration & viability range
Measurements can be made for cells at concentrations within
the 1 x 104to 1 x 107(preferably 5 x 104to 1 x 107) cells/mL

11
www.logosbio.com
range and for cells within the 1-90µm (preferably, 5-60µm)
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.

12
www.logosbio.com
1.2 Product Contents
The LUNA-FL™ Automated Fluorescence Cell Counter (Cat # L20001) includes the following
components.
Component
Quantity
LUNA-FL™ Automated Fluorescence Cell Counter
1 unit
Power cord (including an adapter)
1 unit
PhotonSlide™
1 box of 50 slides
Acridine Orange/Propidium Iodide Stain
2 x 0.5 mL
Trypan Blue Stain, 0.4%
2 x 1 mL
LUNA™ Fluorescence Calibration Beads
0.5 x 1 mL
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.
Note: 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.

13
www.logosbio.com
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
LED
Excitation Wave Length
470 +/- 20 nm (Blue)
Emission Wave Length
525 +/- 25 nm (Green), 600 LP (Red)
Cell Concentration Range
1 x 104 - 1 x 107(optimally 5 x 104- 1 x 107) cells/mL
Cell Diameter Range
1 - 90 (optimally 5 - 60) µm
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 PhotonSlide™ specifications
Material
Poly(methyl methacrylate) (PMMA)
Dimensions (WxDxH)
25 x 75 x 2.4 mm
Chamber Depth
100 µm
Chamber Volume
10 µl
1.3.3 LUNA-FL™ software (version 3.0)
Dual fluorescence cell counting
Bright field cell counting
Yeast counting
GFP transfection assay

14
www.logosbio.com
1.4 Product Description
1.4.1 Front view of the LUNA-FL™ Automated Fluorescence Cell Counter
The front view of the LUNA-FL™ Automated Fluorescence 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 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. Be sure to check the electrical outlet configuration in
your country.
<Rear View>

15
www.logosbio.com
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. The slide port is used to insert the slide loaded
with the sample into the instrument.
<Right Side View>
1.4.4 Left side view of the LUNA-FL™ Automated Fluorescence 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

16
www.logosbio.com
1.4.5 The PhotonSlide™ for the LUNA-FL™
The PhotonSlide™ consists of two chambers, labeled as Aand 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. The counted cell volume is about 0.5 µl, almost the same as five (1 mm
x 1 mm) squares in a standard hemocytometer.
Note: 0.4% Trypan Blue Stain is blue and Acridine Orange/Propidium Iodide Stain is pink (or orange).
Sample loading chamber (marked as A and B)
Sample loading port

17
www.logosbio.com
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 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.
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.

18
www.logosbio.com
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

19
www.logosbio.com
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

20
www.logosbio.com
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