New Chapters from the Molecular Probes Handbooksct.saiblogs.inf.um.es/wp-content/uploads/sites/10/2015/... · 2015-10-09 · Introducing the Attune® NxT Acoustic Focusing Cytometer

1 Proprietary & Confidential

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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

[email protected]

New Chapters from the Molecular Probes® Handbook


The Handbook on Fluorescence Detection

www.lifetechnologies.com/handbook


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


Attune® NxT Acoustic Focusing Cytometer

• Unprecedented

modular design

• Fast detection speed

• Distinctive acquisition

& analysis software

• Convenient size


Flow Cytometer components

Laser

Fluidics Optics

Detectors (PMT)

Electronics/SW


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.


Flow Cytometer components

Laser

Fluidics Optics

Detectors (PMT)

Electronics/SW


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


Acoustic focusing

Prior to wrapping

in sheath

Intensity

Count


distribution

Intensity

Count


distribution

1,000 µL/min 12.5 µL/min

High sample input flow rates allow for more

sample flexibility

Acoustic focusing


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


Acoustic focusing

End-on view of capillary


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


1

3

Three key advantages

• Higher throughput saves time and cells

• Precise alignment more confidence in results

• Higher Sensitivity clearer results


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


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


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.


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


EVOS® Imaging Systems


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


LED Light source

• Shorter light path provides better detection of fluorescence signals


Choice of 15 LED Light Cubes + Custom cubes


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


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.


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.


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


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


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


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


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


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.


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


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








Necrosis vs Apoptosis

Z. Darzynkiewicz, et.al.,Cytometry 27:1–20 (1997)


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


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


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


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.


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


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


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.


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.


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.


FUCCI Cell Cycle Sensor

Atsushi Miyawaki Lab, Riken Institute


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


Click-iT™ EdU Cell Proliferation Assay


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++


47

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


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


Click-iT™ EdU: Mouse Intestine

Pink: Click-iT™ EdU

Blue: DNA


Click-iT™ EdU: Mouse Intestine

Orange: Click-iT™ EdU

Green: DNA


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


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


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.


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


Cellular Lights™ and Organelle Lights™

HeLa cells transduced with Cellular Lights™ Actin-GFP

and Hoechst 33342, viewed at 40×


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.


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

http://www.lifetechnologies.com/flowresources

http://www.lifetechnologies.com/flowcytometry

http://www.facebook.com/cytometry

http://www.lifetechnologies.com/apps


THANK YOU! www.lifetechnologies.com/attune

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Documents

New Chapters from the Molecular Probes Handbooksct.saiblogs.inf.um.es/wp-content/uploads/sites/10/2015/... · 2015-10-09 · Introducing the Attune® NxT Acoustic Focusing Cytometer