VWR avantor 49893-2000 User manual

Instruction manual
VWR®Automated Cell Counter Fluo
EU cat. no 49893-2000
___
Version 01 Issue 03/2023

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Legal address of Manufacturer
Europe
VWR International bv,
Researchpark Haasrode 2020
Geldenaaksebaan 464 B-3001 Leuven
[phone]
be.vwr.com
UK Importer:
VWR International Ltd
Hunter Boulevard, Magna Park
Lutterworth, Leicestershire, LE17 4XN
uk.vwr.com
Country of Origin
China

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Table of Contents
1- SAFETY INFORMATION...............................................................................................................................................4
1.1 GENERAL SAFETY REQUIREMENTS ...........................................................................................................................................4
1.2 ELECTRICAL SAFETY.................................................................................................................................................................4
1.3 CHEMICAL WASTE SAFETY.......................................................................................................................................................4
1.4 BIOSAFETY................................................................................................................................................................................5
1.5 CERTIFICATES...........................................................................................................................................................................5
2- PACKAGE CONTENTS .................................................................................................................................................6
3- INTENDED USE..............................................................................................................................................................6
4- SYMBOLS AND CONVENTIONS ................................................................................................................................ 7
5- PRODUCT SPECIFICATIONS ......................................................................................................................................8
6- INSTRUCTIONS FOR USE............................................................................................................................................9
6.1 OVERVIEW................................................................................................................................................................................9
6.2 DESCRIPTION OF BUTTONS AND SWITCHES .........................................................................................................................9
7- OPERATION.................................................................................................................................................................. 10
7.1 START-UP ............................................................................................................................................................................... 10
7.2 PROFILES................................................................................................................................................................................ 10
7.3 PROFILES LOADING............................................................................................................................................................... 15
7.4 SAMPLE PREPARATION ......................................................................................................................................................... 15
7.5 METHODS OF STAINING........................................................................................................................................................ 16
7.6 LOADING SAMPLES INTO SLIDE.............................................................................................................................................17
7.7 BRIGHT FIELD ASSAYS........................................................................................................................................................... 18
7.8 FLUORESCENCE ASSAYS.......................................................................................................................................................23
7.9 RESULTS STORAGE................................................................................................................................................................26
7.10 PREVIOUS COUNTS .............................................................................................................................................................27
7.11 SYSTEM SETTINGS ................................................................................................................................................................27
8- TROUBLESHOOTING .................................................................................................................................................30
9- GENERAL MAINTENANCE .........................................................................................................................................31
10-USER REPLACEABLE ACCESSORIES AND SPARE PARTS ..............................................................................31
11-TECHNICAL SERVICE.............................................................................................................................................32
12-WARRANTY ..............................................................................................................................................................32
13-COMPLIANCE WITH LOCAL LAWS AND REGULATIONS..............................................................................32
EQUIPMENT DISPOSAL .....................................................................................................................................................33
APPENDIX —OUR LOCAL OFFICES IN EUROPE, AMEA AND NORTH AMERICA................................................34

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1- Safety Information
Please follow the guidelines below, and read this manual in its entirety to ensure safe operation of the
unit.
1.1 General safety requirements
a) Please follow the instructions on all safety warnings and labels in the manual and on the
instrument. The instrument should only be used by professionals within the scope of application,
with proper components and accessories suitable for the instrument to be used. The
manufacturer will not be held liable for any damage to the instrument caused by any improper
operation, including damage to any third party.
b) Users are allowed to maintain the instrument only to the extent of recommended maintenance
stated herein（Refer to Chapter -9 General maintenance）. Any change to the instrument
without the manufacturer’s authorization may cause failure of the instrument.
c) In case the instrument breaks down or requires any service, shut down the instrument and
contact with VWR customer service.
1.2 Electrical safety
The following provisions should be followed for the safe use of the electrical devices accompanied
with the instrument:
a) Never disassemble the instrument without any authorization against any possible electric shock.
b) Check whether the working voltage settings of all components are correct before connecting
the instrument to the mains. Use the power supply compatible with the instrument and see the
model label of the instrument for the compatible power supply.
c) Connect the instrument correctly, and ensure it is properly grounded, and the cell counter need
to be plugged to a conform electric and safe system. Any missing or failure to install the fuse, or
the failure to connect to any ground wire may pose a risk of electric shock.
d) Make sure to clean power cords.
e) If any potential unsafe operation is detected, power off and unplug immediately to disconnect
the mains.
f) If any liquid gets contact with the circuit of the instrument, power off and unplug immediately
to disconnect the mains, and wipe the moistened parts of the instrument dry in a timely manner.
g) Keep the bench always dry during operation.
h) The fuse type (rated voltage, rated current and model) must comply with the manufacturer's
specifications. Do not use any unserviceable or used fuse, and avoid short circuit of the fuse.
i) The instrument must be grounded to prevent accidental electric shock.
1.3 Chemical waste safety
Caution! Hazardous Waste. Please refer to the SDS and relevant local regulations for handling and
disposing of waste.
To minimize the risk of chemical waste, users must:
a) Read and understand the SDS provided by the chemical manufacturer, handling or disposal of
chemical waste.

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b) Provide main and auxiliary containers for the waste storage. (The main container is used to store
direct waste, and the auxiliary container to store waste spilled or leaking from the main
container. Both containers must be compatible with the chemical properties of the waste
materials and comply with local regulations on waste storage.)
c) Minimize exposure to chemicals. Wear appropriate protective articles (such as protective
glasses, gloves or protective clothing) for chemical handling. For detailed safety guidelines,
please refer to SDS.
d) Minimize the risk of inhalation of chemicals. Close the chemical containers after use immediately.
Use chemicals only in a well-ventilated environment (e.g. fume cupboard). For detailed safety
guidelines, please refer to SDS.
e) Handle chemical waste only in a well-ventilated environment.
f) Use the accompanying cover to seal the waste containers emptied.
g) Disposal of waste in trays and bottles in accordance with good laboratory practices and local
environmental and health regulations.
1.4 Biosafety
It is possible that biological samples (such as samples of tissues, body fluids and blood of human
and animals) may transmit infectious diseases. Follow all applicable local regulations from time to
time. Wear suitable protective glasses, clothing and gloves.
1.5 Certificates
Complies with the requirements of the
EC EMC directive 2014/30/EU with amendments.
Test Standards:
EN IEC 61326-1: 2021
EN IEC 61326-2-6: 2021
EN IEC 61000-3-2: 2019/A1: 2021
EN 61000-3-3: 2013/A2: 2021

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2- Package Contents
Configuration
Part numbers
Items
Quantity
Purpose Description
Standard
49893-2000
VWR® Automated Cell
Counter Fluo
1
Cell count
Standard
/
AC adapter (EU)
1
Power supply
Standard
/
AC adapter (UK)
1
Power supply
Standard
10228-0050
Cell counting slide
1
1 box of 50 pcs
Standard
/
USB flash driver
1
Data transfer
Optional
49893-4951
Fluo Cube Green GFP
1
3 fluorescence cubes are
available separately for
selection to provide
excitation light sources of
different wave lengths
Optional
49893-4952
Fluo Cube Red RFP / PI
1
Optional
49893-4950
Fluo Cube Blue DAPI
1
3- Intended use
This equipment is designed for measurement and further fluorescence analysis of cell quantity and
viability for cell lines, stem cells and primary cell samples. For research only. Not intended for
diagnostics use.

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4- Symbols and conventions
Flammable environment hazard
Never operate the instrument in an environment with flammable gases
Electromagnetic interference hazard
Make sure to operate the instrument in a controlled electromagnetic environment to avoid
any risk associated with instrument failure.
Radiation hazard
Follow all applicable radiation safety procedures all the time when handling radioactive
samples. Make sure to take appropriate disinfection and safety measures during disposal of
radioactive pollutants. Operators must always wear protective supplies (such as particle
protective mask, gloves, protective shoe covers) according to the relevant rules and
regulations of the respective laboratories for the disposal of radioactive pollutants which
must be performed in accordance with the relevant regulations.
Biological infection hazard
Samples used in the intended operation of the instrument may be infectious. For this reason,
it is recommended to follow the general laboratory regulations on infection control
procedures. For information on decontamination agent, its use, dilution and effective
application, please refer to the Laboratory Biosafety Manual issued by the World Health
Organization in 2020. Follow all applicable safety procedures all the time when handling
infectious samples. Make sure to take appropriate disinfection and safety measures during
disposal of infectious substances. Operators must always wear protective supplies (such as
particle protective mask, gloves, protective shoe covers) according to the infection control
procedures of the respective laboratories, and infectious pollutants must be disposed of in
accordance with the relevant regulations.

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5- Product Specifications
Parameter
Description
Cell size range
Recommended 7-60 μm, allowable 4-60 μm
Cell circularity range
1-100 (min./max. size)
Cell concentration range
104-107cells/mL
Cell counting time
<9 s
Accuracy of multi-recognition
CV<5%
Built-in memory
1,000 entries
Touch screen resolution
1024*600
Operation response time
100 ms
Optics
5 megapixel camera, 2.5X optical magnification
Fluorescence cube (optional)
DAPI(375/28Ex 460/50Em)
AO/GFP(480/30Ex 535/40Em)
PI(540/25Ex 620/60Em)

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6- Instructions for use
6.1 Overview
In the process of cell culture, it is necessary to determine the cell concentration and identify the cell
viability to guide the subsequent experiments. For some more complicated cell experiments or samples,
fluorescence analysis is needed to distinguish and count the quantity of a certain type of cell. For
example, count the quantity of cells expressing GFP fluorescence in the transfection experiment.
The automated cell counter collects and analyzes cell images and then obtain the concentration
of the sample. After trypan blue staining, the proportion of living/dead cells can be obtained.
Specific fluorescence analysis can be performed after the fluorescence cube is installed.
6.2 Description of Buttons and Switches
Fig. 6-1 Fig. 6-2
①Touch screen display: 7-inch capacitive screen displaying user operation interface.
②POWER Indicator.
③USB port 1: allows users to download the cell count data and image onto an external device with the
USB port for storage. USB flash drive format: FAT32.
④Counting slide port: allows users to insert the counting slide loaded with samples.
⑤USB port 2：the function is the same as USB port 1.
Note: two USB ports are provided at the front and rear of the instrument for data transfer, and the
instrument does not support reading of two USB flash drives simultaneously.
⑥Quick knob on rear cover plate: users are allowed to loosen two quick knobs by counter clockwise
rotation.
⑦Rear cover plate: allows users to use fluorescence cubes (optional) and store fluorescence cube tools.
⑧Power switch: main power switch.
⑨Power input: users must use provided power cord and proper plug in compatible with the socket in
user’s country to connect the instrument to the socket by the power input.
1
2
4
6
7
5
9
8
3

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7- Operation
7.1 Start-up
When the instrument is properly wired, users are allowed to start up the cell counter. Press the power
switch to start up the instrument and enter the main interface as shown in the picture below.
Fig. 7-1
The main interface contains four parts: System Settings, Previous Counts, Profiles, and Count.
7.2 Profiles
In the bright field (BF) assays, users are allowed to set the threshold values of the count results based
on the profile sliders of size, brightness and circularity; in the fluorescence field (FL) assays, users are
allowed to adjust the RFU slider as required to set the threshold values of the count results.
The Profiles interface allows users to adjust the threshold values of the profiles to be used, rename and
save them for calling of such adjusted Profiles in the next cell counting.
Bright field (BF) Mode
Click [Profiles] on the main interface to enter the profiles setting interface, with the instrument in Non-
Trypan Blue Mode (Non-TP) by default under which the total cell concentration, histogram of total cell
size distribution histogram and average size are available for view.
Fig. 7-2

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Click [Trypan Blue] (TP) to switch to Trypan Blue mode under which the total cell and dead/ live cell
concentrations, histograms of dead / live cell size distribution and average size are available for view.
Fig. 7-3
Fluorescence field (FL) Mode
The following figure shows the adjustment of the fluorescence threshold values. The count result in this
mode includes the total cell recognition chart, total cell concentration, AO/GFP, DAPI, PI cell
concentration, total cell size/quantity distribution histogram, fluorescence intensity/cell quantity
distribution histogram, etc.
Note: if the BF channel is only selected on the Count interface in the FL mode, only the total cells are
available for calculation, and fluorescent cell will be undistinguishable.
Fig. 7-4

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•Parameter adjustment
Size, brightness and circularity sliders
Size: drag the slider to enable the system to screen the cells by the size while counting.
= Larger size = Smaller size
Size screening: allowable range of 7-60μm by default, the lower limit of identification up to 4μm;
When there are much debris in the sample, the interference of cell debris can be eliminated by
adjusting the recognized size range and setting the threshold value.
Brightness: drag the slider to enable the system to screen the cells by brightness while counting.
= Brighter target =Dimmer target
Circularity: drag the slider to enable the system to screen the cells by circularity while counting.
=Less circular target = More circular target
The circularity slider can only be used to set a single threshold value; cells within the set value
range will be counted, excluding the cells beyond the range. Drag the slider toward the target to
adjust the circularity threshold.

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RFU：drag the slider to enable the system to screen the cells by RFU while counting.
RFU slider
= Target with higher RFU = Target with lower RFU
RFU ranges from 0 to 255, and in general it is not necessary to make adjustments in the profiles.
After imaging, the device will automatically calculate the RFU of the background, and set the
lower limit of RFU above the background, so as to filter it.
The automatically calculated background can usually be distinguished from the fluorescent cells,
but its threshold value will fail in the event of too strong the fluorescence background or too weak
cell fluorescence, possibly leading to the removal of the weak cell fluorescence or the presence of
residual background fluorescence. Then, the RFU threshold needs to be adjusted to achieve the
best effect of background removal and fluorescent cells retaining.
Fig. 7-5
Too strong fluorescence background leads to residual background
Click or hold down the above icon to adjust each parameter threshold.
Click the [Help] symbol in the upper right corner of the interface to learn more about the
concepts of size, brightness, circularity, RFU and so on as shown in figure 7-6.

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Fig. 7-6
Quick Mode
By checking [Quick Mode], you will perform count in a quick mode; otherwise, you will perform count
in a standard mode. After selecting the count parameters in the Quick mode, once the counting slide
is recognized, the number pad pops up for user to enter a Profile name, and then the system will
count the cells and output the results; in the Standard mode, the focusing results will be presented
first, and then the count results will be output after manually clicking the [Count] key.
The quick mode is suitable for cell samples with no debris and moderate concentration. It can
automatically focus and count after inserting the counting slide, which can greatly reduce the
counting time. Even if the imaging effect in the counting results is not ideal, you can click the back
key to enter the Count interface again for manual fine-tuning.
Note: due to complex fluorescence applications, Quick Mode is not recommended for fluorescence
counting.
Auto Exposure and Auto FL Exposure
Users are free to choose to activate [Auto Exposure] or [Auto FL Exposure], of which the Auto
Exposure features allow users to adjust the BF/FL brightness respectively depending on the
characteristics of cells for count, and then choose whether to restore the system to the default
brightness after counting.
Autosave results
When [Autosave results] is checked, the counting is complete and the counter exits, the data can be
saved automatically.
Note: Autosaving succeeds only when the local storage is sufficient.
Restore to Default Mode
Click [Default] to restore part of the initial settings, including Auto FL Exposure, Auto Exposure, Quick
Mode, Standard Mode, threshold value of each profiles, etc.

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7.3 Profiles Loading
Fig. 7-7
In the left column of the Profiles interface, you can delete/ add/ rename/ Lock profiles by clicking the
icons of respectively.
Note: Locked parameters cannot be edited.
Click lock key to lock the selected profile, the icon is also displayed on the right of the profile
name; if you want to unlock the profile name, select it again,the lock key at the bottom becomes the
unlock key , click the unlock key and confirm as prompted
Here, in addition to the “Default” profiles, up to 17 profiles can be added, which can be called directly
before and during count.
Each profile defines the count parameters (size, brightness, circularity and fluorescence intensity) to
simplify the workflow. After selecting a profile, click [Load] to apply the specified count parameter to
all new cell counts. Users already completing counting, are allowed to apply the new count settings to
the current count results (total cells, viability, etc.) and all new counts by loading new data from the
Results interface.
7.4 Sample Preparation
The instructions below should be followed for the best results:
1) Make sure to mix the cell suspension well before drawing the cell sample. After the cell sample has
been standing for a long time, it is necessary to flick the tube or pipette the cell suspension gently
and draw the sample from the centre of the tube rather than its bottom.
2) Cell concentration range: 1 x 104–1 x 107cells/mL, optimally 1 x 105–4 x 106cells/mL.

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3) Make sure that the count area is covered with cell suspension to get accurate cell viability analysis
results and start counting immediately after staining according to the analytical protocol.
4) Do not press the optical surface of the counting slide, but hold it by the edges.
5) Carefully avoid any bubble formed in the sample.
6) Minimize the amount of debris in the cell sample to the greatest extent. No matter which counting
or staining method is selected, it is difficult to count and assess the viability of samples containing
large amounts of debris which may come from trypan blue stain or cell samples. Possible treatments
for different sources of debris are provided as follows:
Centrifuge trypan blue solution and take the supernatant;
Centrifuge and re-suspense cell suspension.
7.5 Methods of Staining
Tips: If you only need to count the total cell concentration, you could directly load sample without
staining.
⚫Trypan blue staining
Recommended staining method:
1) Mix 10μL cell suspension with 10μL 0.4% Trypan blue solution.
2) After pipette and mixing well, transfer 10μL of stained cells into the cell counting slide for analysis.
•AO/PI Staining
Recommended staining method:
1) Staining according to the recommended method and concentration in the PURCHASED AO/PI kit.
In most cases, when the final concentration of the stain solution is 2-20μg/mL, the cell samples
incubated for 5-30 minutes will produce bright nuclear staining fluorescence. However, in certain
cell types, conditions, and applications, optimization is required. At this point, the concentration of
stain solution can be increased or decreased for the best staining strength.
2) Rapid staining:
Prepare a staining premix of 10μg/mL AO and 50μg/mL PI in advance.
Transfer 20μL cell suspension into tube, and add AO/PI premixed staining solution of the
same volume (the recommended AOPI final concentration is AO: 5μg/mL, PI: 25μg/mL).
After pipette and mixing, incubate for 3-5min.
After pipette and mixing well, transfer 10μL of stained cells into the cell counting slide for
analysis.
•DAPI Staining
Recommended staining method:
Staining according to the recommended method and concentration in the PURCHASED DAPI kit.
In most cases, when the final concentration of the DAPI stain solution was 0.1-1μg/mL, the cell
samples incubated for 3-10 minutes will produce bright nuclear staining fluorescence. However, in
certain cell types, conditions, and applications, optimization is required. At this point, the
concentration of stain solution can be increased or decreased for the best staining strength.
Note: since DAPI stain solution is semi-permeable and can stain both live cells and fixed cells, longer
staining time for live cells is required, otherwise the proportion of stained cells will be lower. For

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avoidance of longer waiting, users are allowed to add 75% alcohol of the same volume to kill the cells
for staining if don't need to count the cell viability.
7.6 Loading Samples into Slide
1) After pipette and mixing well, transfer 10μL of sample into the cell counting slide for analysis.
2) Allow the sample to stand for 30 seconds in the slide, and insert the sample-loaded slide into the
slide port. When correctly inserted, a soft click will sound.
3) After counting, just push the counting slide gently into the slide port until it clicks and is pushed out
under spring pressure. Hold the counting slide by the edges to take it out.
Note: do not reuse the disposable counting slide, which may influence the count results.

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7.7 Bright Field Assays
If the system recognizes insertion of the counting slide, the file name editing interface will pop up.
Shown as figure 7-8.
Fig. 7-8
Enter the file name and click [Enter] or click [skip] (“skip” means that the default name or last typed
name is selected) to enter the count interface. Here you could start counting and perform such actions
as zooming in, fine tuning of focus and light source brightness adjustment, so as to capture optimized
images for accurate count of cell concentration or dead/ live cells.
Count page of TP/Non-TP mode:
Fig. 7-9
in the figure above shows the active profile file name. Click this icon to enter the
Profiles page.
For setting the dilution factor of the sample. Click“1×”to pop up a numeric keypad. The
range of setting value: 1.00~999.00.
Note: The default dilution of Trypan Blue mode is “2×”, and the default dilution of "AO+PI" mode is “2×”.

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Dilution factor of sample:
The cell concentration counted by cell counter is the measured concentration of the cells on the
current counting slide, which may be accessible from the dilution of the original solution; so users need
to convert the current concentration into the concentration of the original solution to calculate the
volume of the original solution required for dilution to the target concentration.
allows users to adjust the light source brightness. If users check [Auto Exposure], the results will be
auto adjustment values by default. Users can also manually adjust the brightness of light source by
clicking & . Adjustment range: 0-100.
Brighter or dimmer effect may have an effect on the imaging quality. In general, when automatic
exposure is activated, better bright field white light illumination can be obtained automatically on the
imaging interface. The overexposed, moderate and underexposed effects of the bright field are as
shown in Fig. 7-10 (A-C) below.
A
B

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C
Fig. 7-10
A. Overexposure: brighter light source, whiter background, and the cell details shaded by light,
affecting cell recognition.
B. Underexposure: dimmer light source, darker background, and darker cells as a whole, affecting
the judgment of the live and the dead cells.
C. Normal exposure: moderate brightness of light source, clear outline of cells, live and dead cells
showing significantly different transmittance characteristics.
Focusing:
When the counting slide is inserted, the system recognizes the counter and starts focusing
automatically.
Click to pop up the slide bar: .
Click [Auto] to execute auto focus.
Click and to adjust the focus. Focus value is displayed in real time, such as "1230" showed
above the bar. Adjustment range of aggregation value: 0-2400.
The mean of [Set]: customize the focus value. The next time the instrument is used, the system will
focus based on the focus value of the last “Set” in order to quickly obtain good pictures for accurate
counting.
Note: the point of set will be influential to autofocus.
Focusing recommendations:
It is critical to ensure proper focus to accurately identify cells and distinguish dead or live cells. Cell
images with correct and incorrect focus are as shown in Figure 7-11(A-B)