Basic Training Invitrogen™ Attune™ NxT Flow Cytometer...The Attune NxT Flow Cytometer with...

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Ryan Chu 朱伯逢, Ph.D(c) | Field Applications ScientistAug 2020, National Defense Medical Center

Basic Training

Attune™ NxT Flow Cytometer

For research use only. Not for use in diagnostic procedures


Introduction to

Flow Cytometry

3

What is Flow Cytometry?

Cyto Metry

Cell Measurement

Measurement of cell properties

Performed using single cell

suspensions

Cells can be measured based on

size, shape, granularity, and light

scattering properties

Fluorochromes are used to label

the cell’s physical properties to

help provide measurements

4

The Flow Cytometer – “A Different kind of Microscope”

Light

Sample

Laser

Data Analysis

Flow cytometry is similar to a microscope.

Microscope produces an image of a cell

A flow cytometer collects and quantifies scattered light and

fluorescence. The output produced is numbers.

5

Green Fluorescence

Red F

luore

scence

Flow Cytometry is more Quantitative than Microscopy

6

Flow Cytometry is more Quantitative than Microscopy

Green Fluorescence

Red F

luore

scence

25% 8.3%

25%41.7%

2. Quantifies amount of

fluorescence

1. Quantifies # of cells with each fluorescence level

3. No subcellular or co-localization information

7

Flow Cytometer Components

1.

2. 3.

4. 5. 6.

8

Applications

• Phenotype Analysis (Cell Surface

Antigens/Markers)

• DNA cell cycle analysis

• Membrane potential

• Ion flux (Calcium)

• Cell viability/Apoptosis

• Intracellular protein staining

• pH changes

• Cell tracking and proliferation (CFSE, KI-67)

• Redox state

• In vivo CTL

• FRET

• Phospho-Flow cytometry

• Total protein

• Fluorescent proteins

• Lipids

• Membrane fusion

• Microparticles

• Phagocytosis

• Enzyme activity

• Oxidative metabolism

• DNA synthesis

• DNA degradation

• Gene expression (RNAFlow)

• Rare events

• The list goes on…If it glows, it

probably flows

9

Flow Cytometry

• Cytometry

Cyto = cell

Metry = measurement

To measure cells…

• Flow

…as they “flow” in a stream of fluid

and pass by a laser

Elements of a Flow Cytometer:

• Fluidics system (Hydrodynamic or Acoustic-assisted hydrodynamic focusing)

• Optics system and Light source-Laser: including PMT detector

• Electronics (converting voltage pulse to electronic signal computer system and

software for data analysis

10

Principles of Flow Cytometry

1. Cells in a single profile pass

through the narrow tube called

flow cell

• Cell Focusing

2. Laser hits individual cell

passing through the flow cell

• Interrogation Point

3. Deflected light hits a series

of detectors

• PMTs

4. The signals from detectors are

interpreted by a computer

• Storage and analysis

Mixture of cells labeled with fluorescent antibodies

Laser

FSC

SSC

11

Principles of Flow Cytometry

Excitation

source

Detectors

Fluidics

Electronics

Size

Complexity

Phenotype

12

Optics system: What Happens to Light When it Hits a Cell?

Laser Light Scatter

• When laser light interacts with a cell, light is scattered in all directions

• The magnitude of the light scatter is dependent on refractive index, size and

complexity of the cells or particles passing by the laser

• Differences in Forward Light Scatter and Side Light Scatter can be used to

distinguish different types of cells or particles

13

Optics system: What Happens to Light When it Hits a Cell?

Laser Light Scatter

• Forward Scattered light (FSC) is impacted by both refractive index and can

sometimes be used as a measure of relative cell size.

• Side-scattered light (SSC) is a measure of cellular complexity, both

surface and internal. SSC is usually collected at 90 degrees to the laser

beam.

Laser

SSC detector

FSC detector

Obscuration Bar

14

Light Scattering in Human White Blood Cells

Lysed human whole blood

Granulocytes

Monocytes

Lymphoid cells

Data

representation

Dual

parameters

representation

Morphology

Dot plot

(size vs complexity)

15

What if multiple Cell Types are with similar Size and Complexity?


Granulocytes

Monocytes

Lymphoid cells

Data

representation

Dual

parameters

representation

Morphology

Dot plot


B cells, T cells, etc.

16

Fluorophore Conjugated Antibodies

Cell surface protein

Intracellular protein

Identify specific cell types Examine protein expression in individual populations

protein

17

What is a flow cytometry antibody?

Primary

antibody Fluorophore

Conjugated antibodies allow for less experimental

steps like washes and centrifugations

Multiple antibodies and reagents are often required

for an experiment to detect the cell population of

interest

Fluorophore is attached to the primary antibody

18

• Fluorophores are compounds that emit light upon excitation

Fluorophores

LASER

DETECTOR

CHANNELS

(PMT)

19

• ORGANIC:

• Contain several aromatic groups

• FITC, PE, Per-CP, APC, eFluors,

Alexa Fluor® , Pacific Blue, Cy5,

Cy5.5, Cy7

• Polymer-dyes

• Super Bright Dyes, Brilliant Violet Dyes™

• Non-ORGANIC:

• Semiconductor particles

• Qdots

Different Fluorophores

FITC

20

Optics of Flow Cytometry

Excitation

source

Detectors

Fluidics

Electronics

Size

Complexity

Phenotype

Optics

21

Collect Precise Range of the Emitted Light Wavelengths

Bandpass Filter Longpass Filter

Shortpass Filter Dichroic Mirror

BP 530/30

460 nm

520 nm

540 nm

700 nm

LP 530

460 nm

520 nm

540 nm

700 nm

SP 530

460 nm

520 nm

540 nm

700 nm

DLP 530

460 nm

520 nm

540 nm

700 nm

22

Flow Cytometry: Detectors

Photomultiplier tube (PMT):

PMT convert photons into electrons and amplify them to

create a voltage pulse. Often referred as the “detector”

Photon

Photoncathode-

layer

Voltage [1-2 kV]

Dete

cti

on

Dynode AnodePrimary Photo-electron

Secondary-electron

Vacuum

23

Sample Presentation: Voltage Pulse

Time

Voltage

Time

Voltage

Time

Voltage

Laser

Laser

Sample flow

Laser

Voltage pulse in PMT

Peak: Height, width, areaLaser

24

Sample Presentation: Voltage Pulse

Time

(microseconds)

PulseArea, A

Volts

Puls

eH

eig

ht,

H

Pulse Width, W0

25

Three Colors Experiment

Sample:

Lysed Blood from Human

Measure:

% T-lymphocytes CD4+

% T-lymphocytes CD8+

T-lymphocytes specific

Antibody Fluorescent Probe

1 Anti-CD3 Alexa Fluor™ 488

2 Anti-CD4 R-PE

3 Anti-CD8R-PE Alexa Fluor 700

tandem dye

26

Three Colors Experiment – Data Representation


Granulocytes

Monocytes

Lymphoid cells

Data

representation

Dual

parameters

representation

Morphology

Dot plot


27

Alexa Fluor 488 fluorescence (CD3)


3 different identifiable regions

(i.e. populations)

1 region of interest (i.e.

lymphoid populations)

One parameter

representation

Fluorescence

Histogram plot

(Fluorescence intensity

vs

cell count)

Data

representation

28



Gating on T-cell population

R-PE fluorescence (CD4)R-PE Alexa Fluor 700

Fluorescence (CD8)

Data

representation

Two histogram plots, one for

each fluorescence

Loss of information!

29

R-P

E A

lexa F

luo

r 700

flu

ore

scen

ce


R-PE fluorescence


Gating on T-cell population

R-PE Alexa Fluor 700

Fluorescence (CD8)

R-P

E A

lexa

Flu

or

700

Flu

ore

sc

en

ce

(C

D8

)

R-PE fluorescence (CD4)

R-PE fluorescence (CD4)

Dual fluorescences Dot plot

Data

representation

30

Spectral Overlap

31

What is spillover and compensation?

BL1 BL2

What is the real R-PE fluorescence?

Subtract AF488 signal in R-PE channel

32

What is Spillover and Compensation?

Un-compensated result

Alexa Fluor 488 is being detected

by the PE channel

Properly compensated result

Alexa Fluor 488 is subtracted

in PE channel

33

Basic Rules of Compensation

• Unstained cells

• Single color controls are required

• Controls need to be at least as bright as the brightest positive sample

• Background fluorescence should be the same for the positive and negative control populations

• Compensation color must be matched to your experimental color (FITC cannot substitute for GFP)

• The actual tandem dye being used in the sample staining must be used in the single color control

• Collect enough events to be statistically relevant


Flow Cytometry

Resources and

Education

35

Fluorescence SpectraViewer & Panel Builder

36

Flow Cytometry Learning Center

Flow Cytometry Learning Center

Learn more about flow cytometry

applications, techniques, and basic

principles.

Molecular Probes™ School of

Fluorescence—Flow Cytometry Basics

Learn how a flow cytometer works including

the fluidics, optics and electronics. This is a

free resource to help you get started with flow

cytometry, which can be a complex and

challenging application.

Flow Cytometry Resource Library

Curated collection of scientific application notes, publications, videos, webinars, and scientific

posters for flow cytometry.

• Flow Cytometry Application Notes, Scientific Posters, and BioProbes Articles Various applications, providing the

conditions and reagents used to achieve the results. Scientific posters presented by our R&D scientists at key

conferences. Articles from BioProbes Journal.

• T Cell Stimulation and Proliferation eLearning Course Modular, animated, and narrated eLearning course on T cell

activation and the methods used to measure T cell function. Knowledge checks and a practical application session.

• Flow Cytometry Educational Videos & Webinars Media for researchers interested in flow cytometry.

• Flow Cytometry Research Tools Fluorescence SpectraViewer, flow cytometry panel design tool, antibodies search

tool, mobile apps and more.

• Flow Cytometry Protocols Step-by-step instructions for successful fluorescence-based assays to measure cell

proliferation, viability, and vitality using your flow cytometer.

• The Molecular Probes™ Handbook—A Guide to Fluorescent Probes and Labeling Technologies Extensive

references and technical notes. Contains 3,000 technology solutions representing a wide range of biomolecular labeling

and detection reagents.

36

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-learning-center.htmlhttps://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/flow-cytometry-basics.htmlhttps://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-learning-center/flow-cytometry-resource-library.html

37

Invitrogen™

eBioscience™ reagents

Antibody

Center

Fluorescence

SpectraViewer

Antibody and Flow Cytometry Experimental Resources

Learn more at:

thermofisher.com/spectra

viewer

Multicolor panel building

Learn more at:

thermofisher.com/flowanti

bodies

Antibody education and

purchase

Learn more at:

thermofisher.com/antibodies

/ebioscience

Immunology antibody

education and purchase

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.htmlhttps://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/antibodies-for-flow-cytometry.htmlhttp://www.thermofisher.com/us/en/home/life-science/antibodies/ebioscience.html?cid=fl-bid-ebioscience


Attune NxT

Flow Cytometer

39

Attune™ NxT Acoustic Focusing Cytometer

40

Attune™ NxT Acoustic Focusing Cytometer

Small in size, big in performance

Footprint (H x W x D):

16 in × 23 in × 17 in

40 cm × 58 cm × 43 cm

Weight:

29 kg (64 lb)

Electrical requirements:

100–240 VAC, 50/60 Hz,

41

The Attune NxT Flow Cytometer with Autosampler

Acoustic-assisted hydrodynamic focusing—

increases sample input speed while maintaining

data integrity

Flat-top lasers—deliver more

even application of light to

each cell

Attune NxT Software—

guides users through

complex experiments

Fluid storage—designed for

minimal waste

Volumetric fluidic system—

provides cell counting and a

resistance to clogging

CytKick (Max)

Autosampler—

one-click transition

from tube to plate


Attune NxT

Fluidics

43

Fluidics

The purpose of a fluidics system is to transport particles in a fluid stream to the laser beam

for interrogation

Only one particle should move

through the laser beam at a time

For optimal illumination, the stream

transporting the particles shouldbe

in the center of the laser beam

Fluidics system needs to be free

of air bubbles and debris

44

Outside Fluidic Components

Fluidics container compartmentSample injection port (SIP)

Status indicator lights

45

Fluidic SIP

Sample injection port (SIP)

Fluid connection ports

for CytKick (Max) Autosampler

Sample injection tube

Sample tube (not included)

Sample tube lifter

46

Fluidics

Focusing fluid

reservoir

Valve

1 mL

sample

syringe

Fluid

lines

Waste Focusing

fluid

Wash

fluid

Shutdown

fluid

Focusing fluid

filters

Sensor connections


Attune NxT

Acoustic Focusing

48

Focused laser

sh

eath

sh

eath

Hydrodynamic core

Focused laser

sh

eath

sh

eath

High sample flow rate

(e.g., 200 µL/min)

Low sample flow rate

(e.g., 12 µL/min)

Intensity

Co

un

t

Broad particle focus =

broad distribution

Intensity

Co

un

t

Narrow particle focus =

narrow distribution

Traditional Hydrodynamic Focusing

Particle positioning in laser is important

49

Acoustic focusing

Prior to wrapping in

sheath

1,000 µL/min12.5 µL/min

Intensity

Co

un

t

Narrow particle focus =

narrow distribution

Intensity

Co

un

t

Broad particle focus =

narrow distribution

High sample input flow rates allow for more sample flexibility

Acoustic Focusing

50

Acoustic Focusing and Hydrodynamic Focusing

Sheath

Hydrodynamic sample core

Focused laser

High sample flow rate

>100 µL/min

Acoustic focusing followed

by hydrodynamic focusing

Intensity

Co

un

t

Narrow data distribution Acoustic-assisted

hydrodynamic focusing

51

Diluting Samples Allows for Quick Sample Input Rate and Lower CVs

Acoustic focusing off Acoustic focusing on Acoustic focusing on

Sam

ple

core

Sam

ple

core

Sam

ple

core

Undiluted

sample

core

Undiluted

sample

core

Diluted

sample

core

52

Acoustic Focusing Capillary

Focused

Particles/CellsCapillary

Piezo-electric device

~20cm Acoustic Waves:

Similar to ultrasound used to visualize a

fetus in utero.

Laser10 µm high

50 µm wide

➢ Sheath fluid is not required to focus cells.

➢ Flow rate can be increased while maintaining resolution.

~ ~~~~ ~ ~ ~~ ~

Flow

53

Acoustic Focusing

End-on view of capillary

54

Comparable Results at All Flow Rates

Traditional Cytometers

µl/min

CV= 4.83 CV=6.12 CV=7.76

12 μL/min 35 μL/min 60 μL/min

Hydrodynamic Focusing Only

12.5 μL/min 25 μL/min 100 μL/min 200 μL/min 500 μL/min 1,000 μL/min

CV=2.99 CV=3.03 CV=2.76 CV=2.94 CV=2.70 CV=2.96

Up to10x Faster than

Traditional Cytometers

Acoustically Enhanced Hydrodynamic Focusing

Attune NxT

µl/min

Cells 14 ul/min 66 ul/min 12 ul/min 120 ul/min 12 ul/min 60 ul/min 500 ul/min 1000 ul/min

Seconds 10,000 42.8 9 50 5 50 10 1.2 0.6

Minutes 100,000 7.1 1.5 8.3 0.8 8.3 1.7 0.2 0.1

Minutes 1,000,000 71.3 15.0 83.3 8.3 83.3 16.7 2.0 1.0

Hours 10,000,000 11.9 2.5 13.9 1.4 13.9 2.8 0.3 0.2

Competitor A Competitor B Competitor C Attune NxT

55

Important Acquisition Guidelines

Sample

flow rateMaximum sample

concentration

1,000 µL/minute 2.1 x 106 cells/mL- Particles >4µm in diameter

- Predominantly acoustic focusing

500 µL/minute 4.2 x 106 cells/mL- Particles >2µm in diameter

- Predominantly acoustic focusing200 µL/minute 6.7 x 106 cells/mL

100 µL/minute 1.3 x 107 cells/mL

25 µL/minute 5.4 x 107 cells/mL- Small particles


Attune NxT

Laser, Optic

& Electronics

57

Lasers & Optics

Coordination of lasers, filters, and detectors to

generate fluorescence signals from a single cell

58

Flat-Top Laser Beam Profile Provides Broader Energy to Cell

Gaussian profile Flattened Gaussian profile

• Stable laser alignment

• Minimized downtime

• Corrects for fluidic instabilities

59

Modular System to Accommodate Multiple Lasers

Color Wavelength (nm) Power (mW)

Violet 405 50

Blue 488 50

Green 532 100

Yellow 561 50

Red 637 100

• From one to four lasers

• Five laser colors available

60

Electronics

Functions of electronics

• Convert detected light signals into proportional electronic signals (voltage pulses)

• Electronic signals are processed by the onboard processor

• Convert electronic signals from the detectors into digital data used for analysis

• Interface with the computer for data transfer

61

Flow Cytometry: Detectors

Photomultiplier tube (PMT):

PMT convert photons into electrons and amplify them to

create a voltage pulse. Often referred as the “detector”

Photon

Photoncathode-

layer

Voltage [1-2 kV]

Dete

cti

on

Dynode AnodePrimary Photo-electron

Secondary-electron

Vacuum

62

BRVY – NEW (B3, R3, V6, Y3) (Cat. No. A29004)

63

RL

2

RL

3

RL1

VL

1V

L2

VL3V

L4

VL5

VL

6

Fibers now

enter from right

side (not from

back of block)

IMPORTANT!

Direction of

light path is

different

Red/Violet block is now on the Right of the instrument

Yellow/Blue block is on

Left side

3 Channels off the Yellow,

2 or 3 off the blue

(configuration dependent)

Optics in V6 system

64

Choosing which channel to use

Laser Excitation (nm) Channel Filter (nm) Common Fluorophores

Violet 405 VL1 450/40 SB436, BV421, Pacific Blue, eFluor 450

VL2 525/50 eFluor 506, Pacific Green

VL3 610/20 SB600, BV605, Pacific Orange

VL4 660/20 SB645, BV650

VL5 710/50 SB702, BV711, Qdot 700

VL6 780/60 BV786

Blue 488 BL1 530/30 Alexa Fluor 488, FITC, GFP, SYTOX Green

BL2 695/40 PerCP-Cy5.5, PI, 7-AAD

Yellow 561 YL1 585/16 Alexa Fluor 568, PE, Qdot 565, PI, SYTOX Orange

YL2 620/15 Alexa Fluor 594, PE-Alexa Fluor 610, PE-Texas Red

YL3 780/60 PE-Alexa Fluor 750, PE-Cy7, Qdot 800

Red 637 RL1 670/14 APC, Alexa Fluor 647, SYTOX Red

RL2 720/30 Alexa 700, APC-Alexa Fluor 700

RL3 780/60 APC-Cy7, APC-Alexa Fluor 750, APC-H7

65

Super Bright Polymer Dyes

66

Performance of SB Dyes

Human PBMC

Mouse bone marrow cells

Human PBMC

67

Super Bright Staining Buffer

68

Attune NxT V6 is Compatible with Complex Panel

The Attune NxT V6 instrument will enable the scientific explorer the ability to expand panel with

ease and reduces time to collect samples with rare populations.

Analysis of cell cycle, cell proliferation, apoptosis (e.g. mitochondrial potential, caspase activation,

membrane asymmetry), etc., can still obtain excellent results from the other Attune NxT model in

the core facility.


Attune NxT

Fluids and Maintenance

70

Fluidic Path

Waste

Flow cell

Laser intercept

points

Movement via syringe pump

Focusing fluid

filter 2

Focusing fluid

reservoir

Focusing fluid

filter 1

Focusing fluid

Movement via pump

Focusing

capillaryAssembly

Sample loop

Bubble sensor

Sample tube

Fluid path Sample path

71

Fluidics Solutions

Attune Focusing Fluid• Buffered, azide-free solution which transports focused

particles to the flow cell for laser interrogation

• Prevents sample from coming into contact with the

walls of the flow cell

• Contains an antimicrobial agent, a preservative and a

detergent designed to minimize bubble formation

Attune Wash Solution

• Solution for removing cellular debris and dyes from the

fluidics system of the instrument

Attune Shutdown Solution

• Solution which minimizes bubble formation and

crystal deposits in the fluidics system when the

instrument is shut down

NOTE: All solutions are provided at 1X

concentration and must be at room

temperature (RT) beforeuse.

72

Bleach Preparation for Decontamination

Bleach is 5.25% sodium

hypochlorite in water. 10%

bleach is a 1 in 10 dilution of

5.25% bleach.

Recommendation

• Prepare fresh bleach

• Use laboratory-grade bleach

• DO NOT USE bleach with additives (such as perfumes or soap)

The final concentration of bleach decontamination solution is 0.5% sodium hypochlorite

73

Fluidics Panel

Service

onlyClick to end any

running routine

Returns unused sample volume

back to the well or the tube

Flushes system between samples

to minimize carryover. Rinses can

be user-initiated or automatic

(every time the SIP is lowered)

Washes the system sample lines and flow cell between

sticky/dirty samples or experiments*

*Requires 10% bleach solution.

Primes the instrument fluidics with Attune focusing fluid

Automatically cleans, sanitizes, and shuts down the

instrument

Clears bubbles from the fluidics lines of the

cytometer with Attune debubble solution

Sanitizes the SIP and sample lines between

sticky/dirty samples or experiments*

Back flush operation to remove clogs from

the sample line

Semi-automated decontamination step

74

Cleaning Functions

Sanitize SIP

Run between users, especially after

sticky samples, DNA stains, or beads

Deep Clean Shutdown

Sanitize system with bleach and

wash solutions for selectable

period of time

System clean and flush with

bleach, wash, and shutdown

Type Cycle Time (min)

Quick 5 10

Standard 15 40

Full 25 60


Quick 5 10

Standard 15 40

Full 25 60


Quick - 1

75

Maintenance Summary

Procedure Frequency

Startup and Shutdown Daily

Visual inspection of sample injection port

(SIP), fluidics tanks and connections, and syringe pumpsDaily

Fluidics maintenance–cleaning functions Daily

Sanitize SIP Between each experiment

Computer maintenance Monthly

Optical filter and mirror inspection Monthly or less

Fluidics decontamination Quarterly

Change focusing fluid filters Quarterly

Syringe replacement Every 6 months or as needed

Deep clean As needed

NOTE: The frequency of maintenance depends on how often you run (or do not run) the instrument.

Experiment Explorer: If you need to scroll through the list of

experiments, it is time to export, back up and delete


System

Maintenance

77

Daily Visual Inspection

Fluidics compartment

• Make sure there are no fluids or salt residues

on the floor of the compartment or around the

connectors or tube junctions.

Check all the fluid levels

• Fill/empty as needed:

• Focusing fluid

• Wash solution

• Shutdown solution

• Waste

Visually inspect the SIP

78

Daily Visual Inspection

• Syringe compartment–confirm there is

no fluid or salt residueon the floor of the

compartment

• Syringe – finger-tighten the syringe; change

if there is a leak or if salt residue builds-up

• Focusing fluid filters – located behind

the wash and shutdown fluidics bottles;

change if thereare any signs of debris/dirt

or if the sample pump stays on too long

79

IMPORTANT

1. Connecting the sensor cable while leaving the fluid line disconnected may result in

increased back pressure and introduction of air into the system.

2. The Attune NxT Flow Cytometer must be idle before refilling the fluidics containers.

3. Do not pull on the lines.

1. Remove the sensor cable from the instrument.

2. Press the metal release buttons to free

the tubing.

3. Fill or empty as needed withroom temperature solutions

Large tanks–1.9 L

Small tanks–175 mL

4. Return to cytometer and reconnect the fluid line, then

the sensor cable.

Filling (or Emptying) Fluid Tanks

80

Daily Instrument Cleaning

Between samples • Rinse–automatically initiated when SIP is lowered, or programmed

for Autosampler

• Sanitize SIP for sticky samples,cell counts

Between users • Sanitize SIP / Sanitize Autosampler SIP (if running plates)–can

use diluted bleach or debubble solution

• Deep Clean for sticky samples (e.g., DNA cell cycle stains; bacteria

samples)

• Three levels: Quick (25 min), Standard (50 min), Thorough (75 min)

End of day • Shutdown (Thorough shutdown preferred)

81

System Decontamination

• Function which facilitates the decontamination of the Attune NxT Flow Cytometer and

the Attune Autosampler fluidics

• Sanitizes the system and fluidics bottles with bleach and wash solutions

• Mostly automatic: 60 min, 3-phase operation with on-screen instructions

NOTE: this function is only available to advanced users and administrators

When?

• As a quarterly maintenance routine to prevent and reduce microbial

growth within the instrument and fluidic bottles

• If the system is likely to be idle for more than two weeks (run it in place of the Shutdown

function)

• If the instrument has been idle for more than twomonths

• Anytime contamination in the fluid lines is suspected–i.e., event rate is too high

• Prior to any service work or shipment for service

82

Waste

container

Focusing fluid

container

Wash fluid

container

Shutdown fluid

container

Potential problem: contamination

• Check tanks for cloudiness or debris in the solutions or brown marks on

the sensor

• Fill the emptied waste container with full-strength bleach** up to the bleach

fill mark (bottom line) on the bottle

**Full-strength bleach

112 mL of conc. bleach (8.25%)

120 mL of ultra bleach (6.15%)

180 mL standard bleach (5.25%)

Contamination

83

Fluidics Replacement Parts

Replacement parts

1.9 L waste tank

1.9 L focusing fluid solution tank

175 mL wash solution tank

175 mL shutdown solution tank

Autosampler focusing fluid solution tank

Autosampler waste tank

Cat. No.

100022156

100022155

100022151

100022154

4477847

4477850

Waste

container

Focusing fluid

container

Wash fluid

container

Shutdown fluid

container

84

Maintenance–Focusing Fluid Filters

• Two filters located behind the wash and shutdown bottles.

• The filters may grow some contamination over time. If discoloration is

evident, replace the filters.

• Replacing focusing fluid filters quarterly reduces the risk of any potential

contamination in the lines.

Focusing fluid filter

Cat. No. 100022587

85

How to Change Focusing Fluid Filters

• Remove shutdown and wash bottles from fluidicscompartment

• Unscrew the top luer fitting

• Unscrew bottom luer fitting and remove filter

• Put fittings on new replacement filter and reattach to unit (bottom leur fitting first)

• Ensure arrow on filter points in the direction of fluid flow (down)

• Prime the filters by running 3X Startup and 2X Debubble followed by 2X Rinse procedures

86

Maintenance–Sample Syringe

• Potential problems

• Check for leaks

• Erratic or no fluid draws up from SIP

• Erratic or no fluid draws up from fluidics tanks

• Cat. No.

1 mL syringe

1 mL Autosampler syringe

100022591

4478686

• Frequency of replacement

• As needed, at least twice a year

87

How to Change the Sample Syringe

• Run Shutdown

• Open the Syringe Pump door located on the left side of the cytometer

• Loosen the knurled thumbscrew below ball end of syringe

• Unscrew top portion of syringe from valve head

• Remove syringe from ball end, pull out and replace with new syringe

• Tighten the syringe 1/4 turn past initial contact of the Teflon insert into the valve for a liquid seal

• Properly seat the ball end of the syringe and tighten the knurled thumbscrew below the ball end

NOTE: No tools should be used to tighten the syringe to the valve

88

Operator/user

Deselect those parameters not needed: minimizefile size

*** Do not clutter the Experiment Explorer ***

Close experiments not currently active (collapse all)

Export then delete experiments from the browser

Export experiments: .atx or .apx

FCS files: 3.0 or 3.1 format

Virus protection–scan thumb drives before connectingto Attune NxT computer

If you need to scroll through the list of experiments in

the Experiment Explorer – it is time to back up and delete

Maintaining Computer Efficiency and Data Quality

89

Database Utility

What does the Attune NxT database utility do?

1. Backup User Data: backs up the entire database, files plus database data, to a folder a

single time (duplicates everything upon initiation)

2. Restore User Data: restores the contents of the database folder from a backup, so that the

backup becomes the current version of the database (e.g., the “live” database)

3. Automated backup: schedules automatic backup of user data and the database

4. Reinstall Database: used to reset the database to the new, no-data added state

(this should rarely need to be used)

• When would this be suitable?

• If recommended by FSE

90

Maintenance Summary

Procedure Frequency

Startup and Shutdown Daily

Visual inspection of sample injection port

(SIP), fluidics tanks and connections, and syringe pumpsDaily

Fluidics maintenance–cleaning functions Daily

Sanitize SIP Between each experiment

Computer maintenance Monthly

Optical filter and mirror inspection Monthly or less

Fluidics decontamination Quarterly

Change focusing fluid filters Quarterly

Syringe replacement Every 6 months or as needed

Deep clean As needed

NOTE: The frequency of maintenance depends on how often you run (or do not run) the instrument.

Experiment Explorer: If you need to scroll through the list of

experiments, it is time to export, back up and delete


Thank you

For Research Use Only. Not for use in diagnostic procedures.

© 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

PerCP-Cy5.5 is a registered trademark of BD Biosciences. Apple is a trademark of Apple, Inc. Android is a trademark of Google Inc.

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