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AttuneTM Acoustic Focusing Cytometer Training
Manik Punj
Attune Training
Attune Training Agenda
Section 1 An Introduction to Flow Cytometry
Section 2 An Introduction to Acoustic Focusing
Hydrodynamic Focusing vs. Acoustic Focusing
Section 3 Instrument Systems
Optics, Fluidics, Electronics
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Break
Section 4 Performance Tracking
Section 5 Software Overview
Ribbons and Menus
What is Flow Cytometry?
CYTOMETRY is the measurement of physical or chemical characteristics of cells or particles
FLOW CYTOMETRY measurements are made as cells or particles in suspension pass individually through a flow cytometer instrument
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cytometer instrument
• Performed on single cell suspensions
• Provides discrete measurements from each cell in the sample
• Provides a distribution of the measured characteristics in the sample
Flow Cytometry: What Can I do?
• Flow cytometric measurement records data from single cells
• Rapid statistical analysis on large numbers of cells are obtained
• Identifying subpopulations within a heterogeneous population
• Ability to identify cell populations on multiple characteristics
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• Ability to identify cell populations on multiple characteristics
• Ideally suited for blood and other cells in suspension
• Ability to archive data
• Data format allows post acquisition analysis
Flow Cytometry Basics
1. Cells in a single profile pass through the flow cell
2. Laser hits individual cellPassing through the narrow tube called flow cell.
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3. Deflected light hits a series of detectors (PMTs)
4. The signal from detectors are interpreted by a computer
Flow cell figure taken from http://www.med.umich.edu/flowcytometry/training/lessons/lesson1/index.htm
Particle Delivery: Hydrodynamic Focusing
Laser Cross Sectional Area
Hydrodynamic core
−narrow particle focus = narrow
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−sh
eath
−sh
eath
−Intensity
−C
ount
−narrow particle focus = narrow distribution
Particle Delivery: Hydrodynamic Focusing
Low Flow Rate High Flow Rate
Laser Cross Sectional Area
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−sh
eath
−sh
eath
−sh
eath
−sh
eath
Particle Delivery: Hydrodynamic Focusing
−C
ount
broad particle focus = broad distribution
Laser Cross Sectional Area
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−sh
eath
−sh
eath
−Intensity
• Increase sample input volume = increase flow rate = decrease in pressure difference = increase core diameter
• Particle distributions broadened
• Instrument resolution decreased
• Volumetric sample rates = 25 ul/min –150 ul/min
Acoustic Focusing:
How does Attune™ Cytometer Differ from Conventional Flow Cytometers?
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What is Acoustic Focusing? A century old phenomenon
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No acoustic force With acoustic force
Acoustic Focusing Capillary
FocusedParticles/cellsCapillary
Piezo-electric device
~20cm acoustic waves – similar to ultrasound used to visualize a fetus in utero.
Laser
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Particles/cellsCapillaryLaser
(~10µm high)
~ ~~~~ ~ ~ ~~ ~
Flow
•Sheath is not required to focus cells•Flow rate can also be increased while maintaining resolution•Flow rate past laser can be precisely controlled to very slow rates,
allowing more fluorescence and scatter signal per particle
Acoustic Focusing High Flow Rate
Laser Cross Section
Low Flow Rate
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Acoustically focused
−sh
eath
−sh
eath
−sh
eath
−sh
eath
PiezoelectricTransducer
Acoustic Focusing Cytometry: Practical Considerations
• Acoustic forces cause cells to line up in the center of the capillary. No sheath flow is necessary for particle alignment.
• Flow rate past laser can be precisely controlled to
Piezoelectric ultrasonic device
CapillaryCells
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• Flow rate past laser can be precisely controlled to very slow rates, allowing more fluorescence and scatter signal per particle (better sensitivity)
• Faster sample flow rates and speedy analysis of dilute samples facilitates rare event analysis
device
Laser(~10µm high)
Detector
Acoustic Focusing
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Acoustically Focused SampleC
ount
−narrow particle focus = narrow distribution
Laser Cross Section
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Acoustic focusing
−sh
eath
−sh
eath
−Intensity
−C
ount
Focus Cells Long Before They Get ThereVariable Laser Interrogation Times
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High sensitivity mode/High sample rate Low sensitivity mode/High sample rate mode
Variable flow speeds
Standard
Laser Interrogation Point Laser Interrogation Point
High SensitivityTransit Time = 40 usec
Total Volume = 600ul/min
Velocity = ~0.5m/s
Standard SensitivityTransit Time = 10 usec
Total Volume = 2400 ul/min
Velocity =~2m/s
Controlling Cell Speed Without Hydrodynamic Focus
High sensitivity Standard sensitivity100 ul/min 100 ul/min0.5 meters/sec 2 meters/sec
FocusingSolution Manifold
Laser
Detector
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Manifold
Cells
Piezoelectric ultrasonic device
Transit Modes and Times
• Standard (2400 ul/min total volume)
Pre-set Sample Input Rate (in ul/min)*
Focusing Fluid Input Rate (in ul/min)
Focusing Fluid to Sample Ratio
25 2375 95:1
100 2300 23:1
200 2200 11:1
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• High Sensitivity (600 ul/min total volume)
500 1900 3.8:1
1000 1400 1.4:1
*note that the particle velocity and interrogation time remains constant regardless of the sample input rate since the total volume (sample and focusing fluid) is constant at this transit mode
Pre-set Sample Input Rate (in ul/min)
Focusing Fluid Input Rate (in ul/min)
Focusing Fluid to Sample Ratio
25 575 23:1
100 500 5:1
Questions ?
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Questions ?
• Flow Cytometer is comprised of 3 subsystems:
• Fluidics - To introduce and focus the cells for interrogation
Section 2 - Instrument Systems
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• Optics - To generate and collect the light signals
• Electronics - To convert the optical signals to proportional electronic signals for computer analysis
The purpose of a fluidics system is to transport pa rticles in a fluid stream to the laser beam for interrogati on.
Three conditions needed for optimal interrogation:
• The stream transporting the particles should pass through the
Fluidics
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• The stream transporting the particles should pass through the focal point of the laser beam.
• Optimally one particle should move through the laser beam at one time.
• Fluidics system needs to be free of air bubbles & debris.
Fluidics
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Attune Focusing Fluid – is an isotonic, buffered, azide-free support/carrier reagent for transporting particles through the capillary assembly to the flow cell for laser interrogation. It contains a preservative and detergent designed to minimize bubble formation. Prevents sample from coming into contact with the walls of the flow cell (optical cuvette).
Attune™ Wash Solution – is a ready-to-use solution for removing cellular debris and dyes from the fluidics system of the instrument.
Fluids
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Attune™ Shutdown Solution – is a 10X solution that prevents bubbleformation in the fluidics system of the instrument. Prepare a 1:10 dilution ofthe shutdown solution in deionized water.
10% Household Bleach Solution in deionized water (0 .5% Sodium hypochlorite) –decontaminates the fluidics lines. Prepare this solution fresh daily and use during the shutdown procedure.
Deionized water – used for diluting Attune™ Shutdown Solution and bleach,as well as for long-term storage of the instrument.
Collection Panel
Run Status indicated the event rate as events per second and the progression of sample collection
Collection criteria allows users to define the endpoint of collection
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Collection controls allows users to run, record, stop sample or clear data
Collection mode allows users to run in high sensitivity mode or standard mode as well as choose sample input rate
Fluidics Functions
• De-bubble is a user-initiated function for clearing bubbles in the fluidics lines of the cytometer.
• Wash is a user-initiated system cleaning between sticky samples. This function requires user supplied 10% bleach solution.
• Unclog function is a user-initiated back flush operation to remove clogs from the sample probe and flow cell.
• Rinse flushes system between samples to minimize carryover. This
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• Rinse flushes system between samples to minimize carryover. This function is run automatically between samples, but it can also be user initiated.
• Shutdown is an automated function that decontaminates, cleans, rinses and powers off the cytometer. This mode requires user supplied bleach, Attune™ Wash Solution, and Attune™ Shutdown Fluid.
• Startup primes the instrument fluidics with Attune™ Focusing Fluid.
• Stop is used to end any running script.
• Clear Error is used to erase any error prompts.
Status Lights
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Routine Maintenance
Procedure Frequency
• Shutdown Daily
• Visual inspection of sample injection port Daily
• Visual inspection of fluidics tanks and connections Daily
• Visual inspection of syringe pumps Daily
• Computer maintenance Weekly
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• Optical filter cleaning Monthly
• Fluidics maintenance As needed*
• Replacing syringes As needed*
• Changing focusing fluid filter As needed*
* The frequency of maintenance depends on how often you run the cytometer. If the Attune is to be un-used for a period of time exceeding two weeks, perform the shutdown function using distilled water.
Daily maintenance
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Daily maintenance
Daily operation - visual inspections
• Fluidics compartment: make sure there is no fluid or salt residue on the floor of the
compartment, around the connectors, or tube junctions.
• Check the fluid levels. Fill/empty as needed
focusing fluid
wash solution
shutdown solution
waste
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• Syringe compartment - make sure there is no fluid or salt residue on the floor of the
compartment
focusing fluid filter – change if there are any signs of debris/dirt or if the
focusing pump stays on for too long. If working correctly, the pump should only be
on seconds/less than a minute.
syringes – change if they leak or have salt residue build-up
Daily operation – startup / shutdown
• Startup Function
• Starts the fluidics, optics and
electronics
• Warms up the lasers (give 15
minutes before running samples)
• Initializes the syringe pumps
• Primes the instrument fluidics
• Shutdown Function
• Turns off lasers
• Cleans and rinses the fluid lines
• Refills fluid lines with shutdown
solution
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• Ensures that:• all fluidic lines are clean
• Syringe pumps are filled with fresh
focusing fluid
• Lasers are warmed up to operating
temperature
• Ensures that:
• Fluid lines are filled with a solution
that prevents crystal formation and
bubbles
• Back up your experiments on a regular basis to a secondary storage device
• Defragment the hard drive of the computer weekly
Regular Computer Maintenance Procedures
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• Minimize memory usage: -When planning the experiments remember to delete parameters that you do not need i.e. only collect the parameters needed
Questions?
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Questions?
Optics
Band Pass FiltersDichroic Mirrors
50mW 405nm Violet Laser
20mW 488 Blue Laser
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Laser Light ScatterForward Scattered light (FS) is proportional to cell-surface area or size.
Side-scattered light (SS) is proportional to cell granularity/internal complexity of the cell. SS is usually collected at 90 degrees to the laser beam.
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.
..
....
.
. .
.Laser
FS detector
SS detector
Fluorescence- Common Definitions
Fluorescence - is the result of a three stage process in certain molecules called fluorophores, or fluorescent dyes.
Absorption spectrum - The wavelength range over which a fluorescent compound can be excited.
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Emission spectrum - The range of emitted wavelengths of a fluorescent compound, it is a longer wavelength than the absorption wavelength.
Auto-Fluorescence - Otherwise know as background fluorescence that originates from endogenous sample constituents or from unbound or nonspecifically bound probes.
This Box is to cover up the word summary and arrow
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Optical Filters
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There are five types of optical filters used in flow cytometry:• Bandpass filter (BP)• Longpass filter (LP)• Shortpass filter (SP)• Dichroic mirror (DM)• Neutral density filter (ND)
Band Pass Filters
Long Pass
Short Pass
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Dichroic Mirror
Laser Channel FilterWavelength
Range
488 nm Blue 20 mW laser
BL1 530/30 551-545 (FITC)BL2 574/26 561-587 (PE)BL3 690/50 665-715BL4 780/60 750-810FSC 488/10 483-493
638 nm Red 50 mW laser
RL1 660/20 650-670RL2 780/60 750-810SSC 638/10 633-643
Product Overview: Attune ® Laser Configuration
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Laser Channel FilterWavelength
Range
488 nm Blue 20 mW laser
BL1 530/30 515-545BL2 574/26 561-588BL3 640LP >640
405 nm Violet 50 mW laser
VL1 450/40 430-470VL2 522/31 507-537VL3 603/48 579-627FSC 405/10 400-410SSC 405/10 400-410
•Compensation is the process by which we correct for "spillover“
•Every fluorescent molecule emits light with a particular spectrum unique to that molecule
•These emission spectra overlap, in some cases very significantly
Compensation
530/3046.8% FITC
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46.8% FITC0.4% R-PE0% PerCP
575/2610.1% FITC53.9% R-PE0% PerCP
648 LP0.1% FITC8.8% R-PE99.7% PerCP
Uncompensated vs. Compensated Data
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Questions?
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Questions?
Electronics
Functions of Electronics:
• Converts detected light signals into electronic signals
• Electronic signals are process by the computer system
• Converts signals from detectors into digital data used for analysis
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Electronics and Computers
Time
Vol
tage
SampleFlow
Laser
Voltage Pulse in PMT
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Time
Vol
tage
Time
Vol
tage
Laser
Laser
PeakHeightWidthArea
Electronic Pulse
Vol
ts
Pulse Area
Pul
se H
eigh
t
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Time(µ Seconds)
Pul
se H
eigh
t
Pulse Width
0
Threshold (“trigger”)
Electronics: Overview
FL2
Time
TimeFSC
FL1
Data
SS
C
FSC
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Time
Time
FL1
SSC
Time
FL3
Data Processor
FL2
Doublets
Doublet Discrimination
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Singlets
Questions?
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• Allows you to monitor performance of the instrument
• Critical to ensure accuracy and sensitivity of instrument
• Includes:
•Running the same bead particle set (AttuneTM Performance Beads)
•Monitoring changes in the CV
Instrument Performance Tracking
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•Monitoring changes in mean fluorescent intensity
•Tracking linearity of instrument
•Evaluating quantum efficiency and background
•Sets laser delay
•Sets the instrument’s performance baseline
• Provides information about all the lasers and detection channels
Attune Performance Tracking Beads• A mixture of beads of four fluorescence emission intensities in
equal concentration− Blank− Dim− Medium− Bright
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• 3mL vial
PN: 4449754
Running Baseline
• Uses performance tracking beads
• CSV file obtained from Applied Biosystems® website
• Performed any time a new lot of AttuneTM Performance beads are used
• Performed after any major maintenance on the instrument
• The percent half-peak coefficient of variation (%HPCV) of the brightest
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• The percent half-peak coefficient of variation (%HPCV) of the brightest bead is recorded
• PMT is adjusted to place the brightest bead at target MFI values, and voltage values for each channel are recorded
• Relative quantum efficiency (rQ) and relative Background (rB) is calculated for each channel
• Linear regression is calculated and recorded
• Laser delay setting is also automatically calculated
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Running Daily Performance Check
• Performed after baseline values have been defined
• AttuneTM Performance Beads are used to run daily performance measurements to track the performance of the cytometer
• Run the performance test at least once per day that the instrument is used
• Determines the voltage required to place the brightest bead in the target MFI, and calculates the delta PMT voltages as compared to the baseline.
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• %HPCV of the bright bead is recorded
• Relative quantum efficiency (rQ) and relative Background (rB) is calculated for each channel
• Linear regression is calculated and recorded
• Laser delay setting is also automatically calculated
• Levy-Jennings charts provides a record of %HPCV and PMT voltage to check for shifts and trends
Daily Performance Check
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Levey-Jennings Reports
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A calibration curve plot showing limit of detection (LOD), limit of
quantification (LOQ), dynamic range, and limit of linearity (LOL).
PMT Voltage
What to do if performance check fails
1. Repeat performance check
2. Check the Performance test troubleshooting section in the users
guide.
3. If additional help is required, don’t hesitate to call .
4. Call: 800-831-6844 or 1-800-327-3002, option 4 (5 am-5 pm PST), or
email: abcc@appliedbiosystems.com
5. The AB Call Center will open a call or simply transferring the call to
the TAC group if assistance is needed.
6. Provide the serial number and contact information
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6. Provide the serial number and contact information
www.appliedbiosystems.com/support
Request instrument repair
Other maintenance
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Other maintenance
Other maintenance - Optics
Filter holders
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• Check for dust or scratches
• Remove any dust from the surface with a blower (compressed gas) or a soft brush.
• If necessary, gently clean using a clean lens cloth and lens cleaning solution or MeOH
• Frequency – monthly or less
Other maintenance optics• Important notes:
− Always allow at least 10 min for the lasers to reach operating temp
− Powering the instrument on/off within 30 minutes can decrease the laser lifetime.
− The “Shutdown” and “Wash scripts” powers off the lasers automatically, allow at
least 10 min for the laser to reach operating temperature
− If you cancel shutdown, allow at least 10 min for the lasers to reach operating
temperature
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Other maintenance – Fluidics decontamination
• Potential problem - contamination− Check for cloudiness or debris
− Discolored solution or brown marks on sensor
• Cleaning instructions: User bulletin # 4468798
Jun 2011
• Frequency: monthly
• Perform ‘system flush’ if the instrument will not
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• Perform ‘system flush’ if the instrument will not
be used for ≥ 2 weeks
• Replacement part #s
- 1L waste # 90039272
- 1L focusing fluid # 90039273
- 250 ml wash soln # 90032053
- 250 ml shutdown soln # 90039274
Other maintenance - focusing fluid filter and syringes
Focusing Fluid Filter
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Syringes/Pumps door10ml Syringe 1ml Syringe
Other maintenance – Focusing fluid filter
Crack
Focusing fluid filter PN 4456564
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� The filters will grow some contamination over time. If you can see brownish red
spot, and if the filter is intact, it should still keep this growth out of the bottle.
� The focusing fluid filter air gap needs to point down ( )
� Replacing focusing fluid filter every 6 months reduce the risk of any potential
contamination in the lines.
� Don’t hesitate to replace focusing fluid filter !
Other maintenance - Syringes
• Potential problem:
− Check for leaks
− Erratic fluid draws from fluidics tanks or SIP
− No fluid draws up from the fluidics tanks or SIP
• Part numbers:
- 1 ml syringe 4452079
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- 1 ml syringe 4452079
- 10 ml syringe 4452819
• Frequency: as needed
Syringe replacement – directions in manual (V 1.2.5)• Run “Shutdown script”. The plunger will be lowered and instrument will turn off
• Open the Pump door and remove the plunger lock screw
• Unscrew the syringe from the valve by rotating it counter-clockwise
• To install a new syringe, pull the plunger down
• Align the syringe with valve and syringe plunger holder assembly
• Rotate clockwise until the syringe end cap seal hits the bottom of the valve
• After bottoming out, rotate clockwise ¼ turn to ensure complete seal
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• After bottoming out, rotate clockwise ¼ turn to ensure complete seal
• Align the hole in the plunger with the hole in the plunger holder assembly
• Insert the plunger lock screw and tighten
Notes:
• No tools should be used to tighten the syringe to the valve.
• Over tightening the syringe beyond the above recommendation could result in
damage to the syringe and/or valve.
• Proper syringe-to-valve seal is crucial for the operation when fluids are cycling
through the system.
Other maintenace: System flush / long term shutdown
• Perform system flush if the Attune will be shutdown for > 2 weeks
− 1. Replace all fluidics container (focusing fluid, wash and shutdown tanks) with
de-ionized water
− 2. Run startup
− 3. Run shutdown function using deionized waster on the SIP instead of bleach
− 4. When shutdown is complete, empty all fluidics tanks and allow to air dry
• This process will prevent crystals from clogging the fluids system.
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• This process will prevent crystals from clogging the fluids system.
Other maintenance: Software • Set up user accounts with operator privileges
• Browser
− Minimize the number of experiments displayed in the browser
− Close all experiments except the one that is currently active (by clicking on the
arrow to minimize experiments)
− Remove experiments from browser but keep data on hard drive
Right click on the experiment name,
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Right click on the experiment name,
Click on delete experiment – the following options are displayed
> Yes – will delete the experiment and associated files
> No – will delete the experiment from the browser and keep the associated files (on
the hard drive)
> Cancel – will make no changes
• Back up your experiments on a regular basis to a secondary storage device
• Check thumb drives for viruses, run Antivirus software to remove threats
• Defragment Hard Drive and run Disk Cleaner (Monthly)
• Minimize memory usage:
Instrument settings - delete parameters not needed i.e. only collect
Other maintenance - regular computer maintenance procedures
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Instrument settings - delete parameters not needed i.e. only collect
the parameters needed
Summary Maintenance
Procedure Frequency
• Start up / Shutdown Daily
• Visual inspection of sample injection port Daily
• Visual inspection of fluidics tanks and connections Daily
• Visual inspection of syringe pumps Daily
• Computer maintenance Weekly
• Optical filter cleaning Monthly
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• Optical filter cleaning Monthly
• Fluidics maintenance Monthly
• Replacing syringes As needed*
• Changing focusing fluid filter As needed*
• The frequency of maintenance depends on how often you run the cytometer.
• If the Attune is to be un-used for a period of time exceeding two weeks, perform the shutdown function
using distilled water.
Software Overview
Main Features:
Tracking and Performance
Experiment Explorer
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Collection Panel
Ribbons
Menus
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.© 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
NOTICE TO PURCHASER: Limited Use Label License
The products shown in this presentation may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the Applied Biosystems website under www.appliedbiosystems.com for the
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Applied Biosystems website under www.appliedbiosystems.com for the comprehensive license information. By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License(s).
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