1 Proprietary & Confidential
The world leader in serving science
Proprietary & Confidential
Introducing the Attune® NxT Acoustic Focusing
Cytometer and EVOS® Cell Imaging Systems
Cell health and apoptosis assays for fluorescent
Imaging and Flow Cytometry
Clara Streiff
Sr. Technical Specialist Flow Cytometry and Imaging
New Chapters from the Molecular Probes® Handbook
2 Proprietary & Confidential
The Handbook on Fluorescence Detection
www.lifetechnologies.com/handbook
3 Proprietary & Confidential
Agenda
Next Generation Cytometry: Attune® NxT Acoustic Focusing Cytometer
Evos ® Cell Imaging Systems
Molecular Probes ® reagents
• Live/Dead™ Fixable Dead Cell Stains
• Cell Event Caspase 3 and 7
• Vybrant DyeCycle™ for live cells
• FxCycle™ Violet and Far Red for fixed cells
• FUCCI Cell Cycle Indicator
• Click-It EDU Cell proliferation
• CellTrace™ Violet Cell Proliferation Kit
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Attune® NxT Acoustic Focusing Cytometer
• Unprecedented
modular design
• Fast detection speed
• Distinctive acquisition
& analysis software
• Convenient size
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Flow Cytometer components
Laser
Fluidics Optics
Detectors (PMT)
Electronics/SW
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Laser/Cell interaction
Forward Scatter: direct laser beam diffraction.
Proportional to cell size
Side Scatter: reflection/rifraction of laser beam
Related to cell complexity ( i.e. organelles) and cell
surface irregularity.
Detected at 90° from laser
Fluorescence: if cell is stained with a probe, or
transfected with a fluorescent protein.
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Flow Cytometer components
Laser
Fluidics Optics
Detectors (PMT)
Electronics/SW
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Focused
laser
she
ath
sh
eath
Hydrodynamic
core
Focused
laser
sh
eath
sh
ea
th
High sample flow rate
(e.g., 200 µL/min)
Low sample flow
rate (e.g., 12 µL/min)
Intensity C
ount
Broad particle focus = Broad
distribution
Intensity
Count
Narrow particle focus = Narrow
distribution
Particle positioning in laser is important
Traditional hydrodynamic focusing
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Acoustic focusing
Prior to wrapping
in sheath
Intensity
Count
Narrow particle focus = Narrow
distribution
Intensity
Count
Narrow particle focus = Narrow
distribution
1,000 µL/min 12.5 µL/min
High sample input flow rates allow for more
sample flexibility
Acoustic focusing
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Hydrodynamic
Sample Core
Focused
laser
High sample flow rate
> 100 µL/min
Intensity
Count
Narrow Data distribution
Acoustic Assisted
Hydrodynamic Focusing
Acoustic focusing and Hydrodynamic focusing
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Acoustic focusing
End-on view of capillary
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Highest Quality LASERs
00.2
0.4
0.6
0.8
11.2
1.4
1.6
x 1
0-4
0 2 4 6 8
10
12
x 1
05
10
010
210
410
610
80
50
100
150
200
250
3001
00
10
210
410
610
80
50
100
150
-1-0
.50
0.5
1
x 1
0-4
-1
-0.5 0
0.5 1
x 1
0-4
CV = 1.1 CV = 1.1
00.2
0.4
0.6
0.8
11.2
1.4
1.6
x 1
0-4
0 2 4 6 8
10
12
x 1
05
10
010
210
410
610
80 5
10
15
20
25
30
35
401
00
10
210
410
610
80 5
10
15
20
25
30
35
40
-1-0
.50
0.5
1
x 1
0-4
-1
-0.5 0
0.5 1
x 1
0-4
CV > 8 CV = 1.7
Mis-Aligned Still Aligned
Customization
• 1 - 4 LASER configurations
• Choose from a variety of LASERs
• Add additional LASERs later
Laser Powers
• Blue:488nm, 50mW
• Violet: 405nm, 50mW
• Red: 637nm, 100mW
• Yellow: 561nm, 50mW
Superior Alignment
• Flat top lasers reduces need for
alignment adjustments
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1
3
Three key advantages
• Higher throughput saves time and cells
• Precise alignment more confidence in results
• Higher Sensitivity clearer results
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No Lyse/No Wash Assays
5µL whole human blood +/- Ab dilute in 3mL PBS/1% BSA acquire
~ 900K events (FSC trigger)
Optional Violet SSC Filter kit available
Trigger with
CD45-Pacific Orange TM
No antibodies
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Comparable Results at all sample rates
• Sample Rates range from 12.5 ul/min to 1mL/min
• 10X faster than traditional systems • 12.5 ul/min (pure hydrodynamic rate) • Up to 1000 ul/min (acoustic + hydrodynamic)
12.5 uL/min 25 uL/min 100 uL/min 200 uL/min 500 uL/min 1000 uL/min
CV=2.99 CV=3.03 CV=2.76 CV=2.94 CV=2.70 CV=2.96
Short acquisition time without loss of data quality
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Attune® AutoSampler
• Reliable mixing while maintaining viability
• Maintains sample purity and data integrity — well carry over <0.5%
• Compatible with standard and deep well 96/384 well plates
• Simultaneous control of tubes or plates
• Easily attached and detached from instrument
• Automated cleaning for easy maintenance.
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Platform Common, Modular Base Platform Architecture
Product
Offering Full Product Line w/7 Laser Configurations
Software 1 Software Version
Manufacturing Base: Single Laser System;
Additional lasers/detectors configured
Autosampler Attune Autosampler
Training Train on a single Platform/Software Version
Service Ease of Manufacture/Service
Attune® NxT Acoustic Cytometer Product Line
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EVOS® Imaging Systems
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Anatomy of an EVOS® Imager
• Large, bright monitor
• Convenient, ergonomic controls
• Fully integrated design
• Interchangeable LED light cubes (up to 4 colors, 15 different light cubes, 50,000 hr
life)
• Adaptable sample holder for almost any sample type
• High resolution camera (monochrome and/or color)
• High quality objectives (from 1.25X to 100X oil immersion)
• Integrated software for data collection and analysis
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LED Light source
• Shorter light path provides better detection of fluorescence signals
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Choice of 15 LED Light Cubes + Custom cubes
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EVOS® Instrument Summary
Slide 22
Descri
pti
on
A
pp
lic
ati
on
XL Core XL FLoid FL/FL Color FL Auto
Basic transmitted
light digital
inverted system
Advanced
transmitted light
digital inverted
system
Basic fluorescence
system
Advanced
fluorescence
system
Fully automated
fluorescence system
Routine cell
culture
In-hood applications
Versatile light
microscopy needs
Chromogenic stains
(ICC, IHC), tissue
culture
Cell Culture
Requiring
Fluorescence
Quick view of cells,
fluorescent labeling
and teaching
Advanced
fluorescence
imaging
Highly configurable
live or fixed
samples
Advanced
automated imaging
Time lapse, Z-stack,
cell counting,
multiwell plate
scanning and image
stitching/ tiling
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EVOS ® FL Auto
Fully automated, multi-channel fluorescence system
• 22” touch-screen LCD display
• Motorized precision X/Y scanning stage, 5 objectives, 4 LED cubes
• Dual cameras (color & B/W)
• Integrated environmental chamber for live cell time-lapse imaging
• Set all acquisition and environmental parameters from microscope
• Precisely maintain physiological or hypoxic conditions
The EVOS® FL Auto Imaging System and
the Onstage Incubator operate as one
fully integrated unit, for an exceptional live
cell imaging experience.
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Z-stacking and Slices
HeLa cells in 12 mm Nunc™ glass base dish transduced with CellLight® Mitochondria-GFP.
Image stacks at 0.12 um spacing collected 100x with Apo objective.
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Image Stitching
Montage of
10x images
20X Scan
Image Stitching: Capture multiple images with overlapping fields and
use mosaic tiling to stitch a high-resolution image of a large area.
SCAN….to get the
big picture
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Mitosis and Proliferation
HeLa cells were transduced with CellLight® Histone 2B-GFP (nucleus,
green) and CellLight® Mitochondria-RFP (mitochondria, red) and imaged
every 30 min over 20 h
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Wound healing Time lapse
Murine glioma cell line, mixed culture of WT and GFP control transfected, DsRed/Cadherin4
ca.21h duration, Phase/GFP/DsRed captured each 10mins, 116 images total
obj: 10x Ph FL
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In the lab
•Excellent phase contrast and brightfield optics with LED light
•Perfect for routine cell culture and maintenance
•High performance optics and patented LED light cube technology
•Outstanding image quality and lowest photo bleaching in the industry
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Agenda
Next Generation Cytometry: Attune® NxT Acoustic Focusing Cytometer
Evos ® Cell Imaging Systems
Molecular Probes ® reagents
• Live/Dead™ Fixable Dead Cell Stains
• Cell Event Caspase 3 and 7
• Vybrant DyeCycle™ for live cells
• FxCycle™ Violet and Far Red for fixed cells
• FUCCI Cell Cycle Indicator
• Click-It EDU Cell proliferation
• CellTrace™ Violet Cell Proliferation Kit
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Alternatives for Dead Cell Identification
• LIVE/DEAD Fixable Dead Cell Stains
• Amine-reactive fluorescent dyes in a range of colors
• Easy discrimination of live and dead cells
Live cells (left) react with the fluorescent reactive dye only on their
surface to yield weakly fluorescent cells.
Cells with compromised membranes (right) react with the dye
throughout their volume, yielding brightly stained cells.
In both cases, the excess reactive dye is subsequently washed away.
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Fixable Dead Cell Stains
Same population discrimination pattern before and after fixation.
Jurkat cells were heat treated (dead) or untreated (live). Cells were
mixed 50% live: 50% dead and stained with LIVE/DEADTM Fixable
Violet Dead Cell Stain.
Before
fixation
After
fixation
Dead
Live Live
Dead
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Fixable Dead Cell Stains
LIVE/DEAD® Fixable Blue stain
LIVE/DEAD® Fixable Violet stain
LIVE/DEAD® Fixable Aqua stain
LIVE/DEAD® Fixable Yellow stain
LIVE/DEAD® Fixable Green stain
LIVE/DEAD® Fixable Red stain
LIVE/DEAD® Fixable Far Red stain
LIVE/DEAD® Fixable Near IR stain
Before Fixation 18 Hours Post-Fixation Before Fixation 18 Hours Post-Fixation
UV (355 nm) excitation, 450/50 filter
405 nm excitation, 450/20 filter
405 nm excitation, 530/30 filter
405 nm excitation, 575/25 filter
488 nm excitation, 530/30 filter
488 nm excitation, 610/20 filter
633 nm excitation, 660/20 filter
633 nm excitation, 780/60 filter
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Necrosis vs Apoptosis
Z. Darzynkiewicz, et.al.,Cytometry 27:1–20 (1997)
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CellEvent™ Caspase-3/7 Green Detection Reagent for the detection of activated caspase 3/7
-A four amino peptide (DEVD) conjugated to a nucleic acid binding dye
-Cell Permeant, intrinsically non-fluorescent
-With activation of caspase-3 or caspase-7 the DEVD peptide is cleaved, enabling the dye to
bind to DNA producing a green fluorescence
NO WASH / NO Background
Control Treated
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CellEvent™ Caspase 3/7 Green Specificity and Robustness
01020
30
40
50
60
70
80
90
NoWash
1Wash
2Wash
3Wash
4Wash
CellEvent™ signal is absent in cells treated with Z-DEVD-FMK caspase inhibitor
% P
ositive A
popt
otic C
ells
Fragile apoptotic cells are
easily lost by plate washes,
introducing assay variance
and error
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Multiplex Time Lapse Imaging of Apoptosis and Mitochondrial Health: CellEvent™ Caspase 3/7 Green and TMRM
Red: TMRM mitochondrial
membrane potential indicator
Fades with apoptosis
Green: CellEvent™ Caspase 3/7
Fluorogenic with
apoptosis
HeLa cells, 0-7 hrs treatment with 0.5 mM staurosporine
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Cell Cycle in brief
G0
M
G2
S
G0: Non-proliferating cells
G1: Period of cell growth where a cell has 2N nuclear DNA content
(2 copies of nuclear genome)
S: Nuclear DNA content doubles to 4N
G2: Cells are maintained at 4N
M: Cell division resulting in two daughter cells each with 2N nuclear DNA
content
Cell cycle describes the progression of a
cell through a cycle of division.
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Cell Permeant Nucleic Acid Dyes
• Dyes which have the ability to penetrate an intact cell
membrane to stain nucleic acid
• These dyes can be used for determining the DNA content of
viable cells.
• Allows resolution of cell cycle information against the
dynamic background of LIVING cells
• Hoechst dyes (UV ex) dsDNA
• Vybrant® DyeCycle™ Violet stain (UV, 405 ex) dsDNA
• Vybrant® DyeCycle™ Green stain (488 ex) dsDNA
• Vybrant® DyeCycle™ Orange stain (488 & 532 ex) dsDNA
• Vybrant® DyeCycle™ Ruby stain (488 & 633 ex) dsDNA
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Vybrant® DyeCycle™ Violet Stain
Staining procedure:
• Resuspend cells
• Add dye
• Incubate 37ºC / 30min.
• Analyze with 405 nm excitation &
450/50 BP
G1
S G2
Channels (440/40 BP (VIOLET)-A)0 40 80 120 160
Nu
mb
er
040
080
012
0016
00
ModFit LT™ Analysis:
DyeCycle™ Violet 5 mM
Jurkat cells in RPMI/10%FBS
Diploid: 100.00 %
Dip G1: 49.28 % at 69.35
Dip G2: 9.41 % at 137.97
Dip S: 41.31 % G2/G1: 1.99
%CV: 4.46
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Side Population Analysis – Vybrant® DyeCycle™ Violet
• Vybrant® DyeCycle™ Violet Stain used for stem cell side populations.
• Same results as Hoechst 33342
• Telford, William G. et al. 2007. Side Population Analysis Using a Violet-Excited Cell Permeable DNA
Binding Dye. Stem Cell 25: 1029-1036.
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FxCycle™ Stains: for DNA analysis in fixed cells
FxCycleTM Violet fluorescence
Count
•Violet 405 nm excitation
•450/50 bandpass filter
FxCycleTM Far Red fluorescence
•Red 633 nm excitation
•660/20 bandpass filter
FxCycleTM Violet stain FxCycleTM Far Red stain
Enables the addition of DNA content measurements to multicolor experiments.
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Premo™ FUCCI Cell Cycle Sensor
• Fluorescent Ubiquitination-based Cell Cycle Indicator
− A fluorescent protein-based sensor using a red (RFP) and a green (GFP) fluorescent
protein fused to different regulators of the cell cycle: Cdt1 and geminin
− In G1 phase, geminin is broken down and only Cdt1 tagged with RFP may be visualized.
Cells in the G1 phase have red fluorescent nuclei.
− In S, G2, and M phases, Cdt1 is degraded and only geminin tagged with GFP remains.
Cells in these phases have green fluorescent nuclei.
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FUCCI Cell Cycle Sensor
Atsushi Miyawaki Lab, Riken Institute
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Proliferation: Measuring DNA Synthesis - Phases of the Cell Cycle
Chronology:
• Detection and measurement of newly synthesized DNA in cells began in the
1960s with the incorporation of radioactive nucleotides (3H-thymidine).
• This was replaced by antibody-based detection of the nucleoside analog bromo-
deoxyuridine (BrdU).
5-Bromo-2’-deoxyuridine
BrdU
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Click-iT™ EdU Cell Proliferation Assay
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46
Detection of Nucleoside analogs of thymidine: BrdU VS EDU
5-Ethynyl-2’-
deoxyuridine
(EdU)
BrdU H
Click chemistry-based detection: Copper-catalyzed azide-alkyne cycloaddition
Azide
Alkyne
+
Alexa Fluor® 488 Dye
DNA DNA
+
Triazole
Cu++
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Click-iT™ EdU Protocol
Click-iT™ detection reaction 30 minutes
Wash 2X 10 minutes
Nuclear counterstain 15 minutes
Wash 3X 15 minutes
Image TOTAL TIME
70 minutes
Incubate with EdU or BrdU, fix & permeabilize sample
With Click-iT™ EdU
• Measure proliferation in cells or tissue
• Time to complete: <2 hours
• Detect by fluorescence microscopy, flow
cytometry or high-throughput imaging (HCS)
BrdU Protocol
Wash 3X 15 minutes
HCl Denaturation 40 minutes
Neutralize 12 minutes
Wash 3X 15 minutes
Block 60 minutes
Anti-BrdU incubation 1-16 hours
Wash 3X 15 minutes
Secondary antibody incubation 120 minutes
Wash 3X 15 minutes
Nuclear counterstain 15 minutes
Wash 3X 15 minutes
Image TOTAL TIME 6-
21 hours
Click-iT™ EdU vs. BrdU Workflow
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Click-iT™ EdU enables content-rich results on HCS platforms,
currently not possible with BrdU.
A B C D
Click-iT™ EdU: High Content Screening and Imaging
A. Newly synthesized DNA with Click-iT™ EdU, B. Cell cycle with Hoechst 33342, C)
Tubulin, D) Composite image
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Click-iT™ EdU: Mouse Intestine
Pink: Click-iT™ EdU
Blue: DNA
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Click-iT™ EdU: Mouse Intestine
Orange: Click-iT™ EdU
Green: DNA
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Cell Proliferation Analysis by Dye Dilution
• Cell division results in equal partitioning of dye between daughters cells.
• Fluorescence of daughter cells is half that of parent cell
First
Generation
Second
Generation
Third
Generation
Fourth
Generation
Brightness
Nu
mb
er
of
Ce
lls
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Distinct Peaks for Each Generation of Cells
100
101
102
103
104
105
0
14
27
41
54
Num
ber
of cells
counte
d
CellTrace™ Violet Fluorescence
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CellTrace™ Violet Analyzed with Proliferation Modeling Software
Human CD8+ T lymphocytes stained with 10µM CellTrace™
Violet and incubated in OpTmizer T-cell Expansion Medium at
37°C for 7 days. (A) Unstimulated cells. (B) Cells stimulated
with 200ng mouse anti-human CD3 antibody and 100ng
Interleukin-2 per milliliter cells.
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CellTrace™ Violet Experimental Protocol
1. Bring a vial of CellTrace™ Violet to room
temperature.
2. Add 20µL anhydrous DMSO to prepare a 5mM
stock solution.
3. Add 1µL of stock solution to 1mL cells for a final
concentration of 5µM.
4. Incubate 30 minutes.
5. Quench and wash.
6. Proceed with stimulation and analysis.
CellTrace™
Violet (Dry)
DMSO
20µL DMSO
5mM CellTrace™
Violet in DMSO
1µL dye into 1mL
cells
Incubate
30min
Quench and
wash Stimulate and
analyze
Dissolve
55 Proprietary & Confidential | Life Technologies Proprietary & Confidential | 7/17/2015
The BacMam system uses a modified insect cell virus (baculovirus) as
a vehicle to efficiently deliver and express genes in mammalian cells
with minimum effort and toxicity.
• BacMam particles are taken up by endocytosis
• Particles migrate to the nucleus
• DNA released for transcription and expression
• Gene expression begins within 4-6 hours of
transduction
• Full expression typically seen after overnight
incubation.
BacMam Basics: Content and Delivery
BacMam: Insect BACulovirus with MAMmalian
promoter
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Cellular Lights™ and Organelle Lights™
HeLa cells transduced with Cellular Lights™ Actin-GFP
and Hoechst 33342, viewed at 40×
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BacMam Talin GFP + Cell Trace Violet
U2OS human osteosarcoma cells transduced with Cellular Lights® Talin-GFP and stained 20 minutes at room temperature with a 5µM solution of CellTrace™ Violet in phosphate-buffered saline.
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Flow Cytometry Resource Center
• Flow Cytometry University
• On-demand webinars
• Fluorescence SpectraViewer
• Posters, application notes, and more
www.lifetechnologies.com/flowresources
Flow Cytometry Mobile App
• Larger selection of protocols
• Simplify your flow cytometric analysis
• Built-in protocol timer and alarm for timed steps
• Available for iPad®, iPhone®, and Android™ devices
Share and learn on Facebook www.facebook.com/cytometry
Flow cytometry resources
www.lifetechnologies.com/apps
59 Proprietary & Confidential
THANK YOU! www.lifetechnologies.com/attune
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