● We define Live Content Imaging as the acquisition, analysis and quantification of images (phase and fluorescence) from living cells that remain unperturbed by the detection method, allowing for repeated measures over long periods of time (days to weeks)

● We differentiate Live Content from High Content Imaging which typically measures assay end points using fixed cells, or employs conditions (e.g. Ab labelling) under which cells are viable for only short periods of time (minutes to hours). Live Content Imaging offers clear advantages for measuring long term biological processes, providing full temporal resolution of the events of interest from viable healthy cells. The images and time-lapse movies are information rich and yield valuable confirmation of the experimental outcomes.

● Here we describe a novel suite of tools – reader technology, cellular reagents, assay protocols, software modules – that together enable true live content imaging assays. The building blocks of these assays are (1) the IncuCyte Zoom live cell imaging device (2) novel, highly validated targeted GFP/RFP lentiviral and stable cell lines (3) sophisticated algorithms for fluorescent object analysis and for quantifying phase structures. Using these tools we have configured micro-titre plate-based kinetic assays for apoptosis, cell proliferation, cytotoxicity, angiogenesis, cell migration, cell invasion and neurite outgrowth. Simultaneous phase contrast and single colour fluorescence assays as well as multiplexed two colour (red/green) and phase reads are exemplified in both homogeneous cell systems (e.g. 1 cell type) as well as co-culture (2 cell types) models.

● The introduction of targeted GFP and RFP cellular reagents suitable for long term live cell imaging along with the 2 colour IncuCyte Zoom system, provide a powerful integrated solution for fully kinetic, multiplexed live cell assays. We foresee particular utility in co- and multi-culture cell systems such as in studies on the tumour microenvironment.

Essen BioScience, Ann Arbor, Michigan, 48108 USA & Welwyn Garden City, AL7 3AX UK

Poster contacts: D.J. Trezise (trezise@essenbio.com) & T.J. Dale (dale@essenbio.com)

SUMMARY

www.essenbioscience.com

live content imaging enabled

Multiplexed, live content cellular imaging enabled: Cell Player

TM reagents, assays and IncuCyte Zoom

TM

enabled

TOOLS, REAGENTS, ASSAYS

Targeted GFP & RFP lentiviruses

3rd generation HIV-based, VSV-G pseudotyped lentiviral particles encoding cytolasmic (CytoLight) or nuclear restricted (NucLight) GFP or RFP, or a combination of the two (DuoLight).

Expression driven off either an EF-1a or CMV promotor with antibiotic resistance cassette for stable cell line generation.

Validated as non-perturbing to cell health across a range of MOIs.

Nuclear / Cytoplasmic GFP / RFP stable cell lines

Created by transduction of the host cell with the targeted lentiviruses (above).

Typically >95% of cells express the fluorescent protein. Validated as comparable to host cell lines (morphology, growth rates, migration rates).

IncuCyte Zoom Live Cell Imaging Device resides within a

standard cell culture incubator.

Cell culture consumables (e.g. micro-titre plates, T-flasks, petri dishes) are placed within for in situ assays.

Gathers time lapse images from cells – high definition phase contrast, green and red fluorescence.

User Interchangeable objectives – 4x, 10x, 20x.

Up to 6 x 384-well plates simultaneously.

Simple to use but highly advanced phase and fluorescent image processing software tools.

HT-1080 NucLight-Red A549 NucLight-Green

VALIDATION OF TARGETTED GFP/RFP

LENTIVIRAL REAGENTS & CELL LINES

PHASE/2-COLOUR ASSAY APPLICATIONS

Co-Culture Kinetic Proliferation

Duplex Cell Proliferation & Apoptosis/Cytotoxicity

2-COLOUR ADVANCED BIOLOGY MODELS

NeuroTrack: Kinetic Neurite Outgrowth

Assay Type Measurement Format

Proliferation Number of nuclei (count) 96, 384w

ApoptosisCaspase 3/7 positive nuclei

(count)96, 384w

CytotoxicityYoYo1 positive nuclei

(count)96, 384w

Migration Scratch wound migration 96w

Invasion

Scratch wound through

substrate (e.g. Matrigel,

Collagen-1)

96w

Angiogenesis Vascular Tube Formation 96w

Neurotrack Neurite Outgrowth 96w

Cell Player Assays

GFP-labelled

HUVECs/ECFCs

Phase (& Fluoresence)

Markers

Nuclear-restricted

GFP/RFP: NucLight

Red/NucLight Green

Bifunctional DEVD/DNA

binding fluorescent

substrate

YoYo-1 cell intact cell

impermeant DNA

Wound confluence (Phase)

Wound confluence (Phase)

>20, including

immortalised and

primary cells

>20, including

immortalised and

primary cells

>20, including

immortalised and

primary cells

>20, including

immortalised and

primary cells

HUVECs & human

dermal fibroblasts

(Prime Kit), Adipocyte

Derived Stem Cells &

Rat hippocampal

neurones, neuronal

IPSCs, neuroblastoma

(Neuro-2a)

Cell types

>20, including

immortalised and

primary cells

3000 nM

1000 nM

333 nM

111 nM

37 nM

12 nM

4 nM

untreated

Figure 1. Lentiviral infection of immortalised and primary cells. Transduction efficiency of NucLight Green at different multiplicities of infection (MOI) ± polybrene in A549 (A) and HUVEC (B) cells following 24-48h transduction. Image of A549 cells expressing the NucLight Green (C). Note the homogeneous nuclear restricted GFP label and healthy appearance of the cells.

96- & 384-WELL KINETIC PROLIFERATION

ASSAYS BASED ON CELL COUNT

Cat# 4490 HeLa NucLight Green

Cat# 4486 HT-1080 NucLight Green

Cat# 4492 A549 NucLight Green

Cat# 4489 HeLa NucLight Red

Cat# 4487 MDA-MB-231 NucLight Red

Cat# 4485 HT-1080 NucLight Red

Cat# 4491 A549 NucLight Red

MOI=

0

MOI=

0.8

MOI=

2.4

MOI=

7.20

20

40

60

80

100- polybrene

+ polybrene (8 g/ml)

% T

ran

sdu

ced

at

48

h

MOI=

0

MOI=

1

MOI=

3

MOI=

6

MOI=

9

MOI=

120

20

40

60

80

100

% T

ran

sdu

ced

at

72

h

A A549 cells B HUVEC C

0 2000 4000 60000

2000

4000

6000

Seeding density (cells/well)

Ob

jec

t c

ou

nt

(1/m

m2)

0 10 20 30 400

250

500

750

1000

Time (h)

Ob

jec

t c

ou

nt

(1/m

m2)

B cell number vs time C log cell number vs time A cell number vs seeding density

Figure 3. (A) Correlation between cell seeding density and nuclear count (HT1080 NucLight-Green stable cell line). (B) Time-course of cell proliferation at different initial cell densities. (C) Log10 of the nuclear count time-course, illustrating exponential cell growth. (D) Kinetic data were fitted to y = y0. eKt to yield comparative growth rate constants (K values) and doubling times.

800

1600

3200

4800

Cells/well

0 10 20 30 4010

100

1000

Time (h)

Ob

jec

t c

ou

nt

(1/m

m2)

800

1600

3200 4800

Cells/well

R2=0.99 Slope 1.0

Mean data from duplicate testing

TAME

-8 -6 -40

1000

2000

3000

4000PD98059

-8 -6 -4

Compound 401

-8 -6 -4

10-DEBC

-8 -6 -4

FAK inhibitor 14

-8 -6 -4

FPA124

-9 -7 -5

KU0063794

-10 -8 -60

1000

2000

3000

4000Cycloheximide

-10 -8 -6

Chrysin

-7 -5 -3

Mitomysin C

-8 -6 -4

Staurosporin

-10 -8 -6

RITA

-8 -6 -4

Doxorubicin

-8 -6 -40

1000

2000

3000

4000Cisplatin

-8 -6 -4

Camptothecin

-8 -6 -4

PAC1

-11 -9 -7

Figure 4: (A) 96-well plate view of kinetic cell proliferation assay in HT1080-NucLight Green cells in the presence of different concentrations of cyclohexamide (CHA). (B) Overlaid timecourses. (C) 384-well assay plate view and IC50 curves.

Cat# 4488 MDA-MB-231 NucLight Green

Figure 2. Panel of NucLight Green and NucLight Red stable cell lines in different host cell backgrounds. Note (1) the discrete nuclear localisation of the fluorescent protein (2) the homogeneous expression of almost all cells in the field of view and (3) the healthy appearance of the cells. In cell proliferation & migration experiments no differences were observed between the properties of the parental and transfected cells (data not shown).

A B

Seeding density

(Cells mm-2)Y0 K Doubling Time (h) R² Y0 K Doubling Time (h) R²

4.8 152 0.045 15.5 1.00 294 0.021 33.6 0.98

3.2 119 0.045 15.5 1.00 222 0.023 30.8 0.99

1.6 63 0.037 18.6 1.00 93 0.025 27.2 1.00

0.8 32 0.032 21.7 1.00 42 0.024 28.5 1.00

HT1080 HelaD

Angiogenesis: Endothelial and stromal cell co-cultures

Cell invasion: HT1080 and MCF-7 cells

Figure 5: (A) Co-culture of HT1080-NucLight-Red and A549 NucLight-Green at 12 h post-seeding. (B) IncuCyte software image mask independently identifying red and green nuclei. (C) Time-course of cell count.

C

Max

ob

ject

co

un

t (1

/mm

2 )

Log [compound] (M)

HeLa NucLight-Red Control (Untreated)

Staurosporin, 300 nM

Figure 6: Cell proliferation and apoptosis. (A & B) Representative images of HeLa NucLight-Red cells following control or 300 nM SSP treatment. Time-courses for nuclear count (C) and caspase 3/7 (D). (E) AUC XC50 curves for caspase 3/7 and nuclear count.

0 10 20 30 40 50

0

200

400

600

1000 nM

333 nM

111 nM

37 nM

12 nM

4.1 nM

1.4 nM

Control

Time (h)

Ob

jec

t C

ou

nt

(1/m

m2)

0 10 20 30 40 50

0

200

400

600

1000 nM

333 nM

111 nM

37 nM

12 nM

4.1 nM

1.4 nM

Control

Time (h)

Ob

jec

t C

ou

nt

(1/m

m2)

A

B

C

D

E

-9 -8 -7 -60

5

10

15

10

15

20Nuclear Objects

Caspase Objects

ProliferationIC50 96 nM

ApoptosisEC50 208 nM

[Staurosporine] M

Ca

sp

as

e 3

/7 (

AU

C x

10

3)

Nu

cle

ar C

ou

nt (A

UC

x1

03)

B C

HT1080 NucLight Red

Figure 7: Cell proliferation and cytotoxicity. (A & B) Representative images of HT1080 NucLight-Red cells following control or 300 nM CMP treatment. Time-courses for nuclear count (C) and YOYO-1 (D). (E) AUC XC50 curves for YOYO-1 and nuclear count.

A

B

C

D

E Control (Untreated)

Camptothecin, 444 nM

0 10 20 30 40 500

20

40

60

804000 nM

1333 nM

444 nM

148 nM

49 nM

16 nM

5 nM

Control

Time (h)

Ob

jec

t C

ou

nt

(1/m

m2)

Nuclear count (proliferation)

Caspase 3/7 (apoptosis)

YOYO-1 (cytotoxicity)

Nuclear count (proliferation)

-9 -8 -7 -6 -50

5

10

15

20

0.0

0.5

1.0

1.5

2.0YOYO-1 Count

Nuclear Count

Proliferation

IC50 17 nM

Cytotoxicity

EC50 197 nM

Log [camptothecin] M

Nu

cle

ar

Co

un

t (A

UC

x1

03)

YO

YO

-1 (A

UC

x 1

03)

A

Ob

jec

t c

ou

nt

(1/m

m2)

Time (h)

Time (h)

Ob

jec

t c

ou

nt

(1/m

m2)

Figure 9: Use of the DuoLight construct for combined nuclear (Red) and cytoplasmic (Green) labelling. This approach allows the use of quantitative algorithms to measure the vascular tube network and HUVEC cell number simultaneously.

Figure 10: Co-culture cell invasion assay in matrigel. Representative images of HT1080 CytoLight-Red and MCF-7 CytoLight-Green cells. Note the invasion of the HT1080 cells, but not the MCF-7 cells into the wounded area.

Figure 8: (A) Representative image of Neuro-2A cells. (B) Neuro-2A cells with the quantification mask applied, identifying neurite outgrowth. (C) Time-course of neurite outgrowth and attenuation by the PKC inhibitor Ro-31-8220.

A B C

0 50 100

0.00

0.05

0.10

0.15

0.20

Control

10 M

1 M

0.1 M

0.01 M

0.001 M

Time (h)

ne

uri

te le

ng

th/o

bje

ct

DuoLight StemKit (ECFCs, ADSCs) PrimeKit (HUVEC, NHDF)

Catalog Name Type Localization Promoter Selection

4475 NucLight Green Lentivirus Nucleus EF-1a Puromycin

4476 NucLight Red Lentivirus Nucleus EF-1a Puromycin

4477 NucLight Green Lentivirus Nucleus EF-1a Bleomycin

4478 NucLight Red Lentivirus Nucleus EF-1a Bleomycin

4481 CytoLight Green Lentivirus Cytoplasm EF-1a Puromycin

4482 CytoLight Red Lentivirus Cytoplasm EF-1a Puromycin

4483 CytoLight Green Lentivirus Cytoplasm EF-1a Bleomycin

4484 CytoLight Red Lentivirus Cytoplasm EF-1a Bleomycin

4413 CytoLight Green Lentivirus Cytoplasm CMV None

TBD DuoLight (Red/Green) Lentivirus Nuc + Cyto CMV Puromycin

Lentiviral targeted GFP & RPFs

Catalog No. Cell Type

4485 HT-1080

4486 HT-1080

4487 MDA-MB-231

4488 MDA-MB-231

4489 HeLa

4490 HeLa

4491 A549

4492 A549

4506 HUVEC (Primary cells)

4511 Neuro-2a

4512 Neuro-2a

TBD HUVEC-DuoLight

NucLight Green Puromycin

NucLight Red Puromycin

Stable cell lines expressing targeted GFP & RPFs

Fluorescent Marker Selection

NucLight Red Puromycin

NucLight Red Puromycin

NucLight Green Puromycin

NucLight Red Puromycin

NucLight Green Puromycin

NucLight Red Puromycin

NucLight Green Puromycin

NucLight Green None

NucLight Green Puromycin

NucLight Red/CytoLight Green None

0 10 20 30 40 500

200

400

600

800

10004000 nM

1333 nM

444 nM

148 nM

49 nM

16 nM

5 nM

Control

Time (h)

Ob

jec

t C

ou

nt

(1/m

m2)

The data contained in this poster represents work designed and conducted by the entire Essen BioScience R&D team

Nuclear Objects

YOYO-1 Objects