1

| Life Technologies Proprietary & Confidential |

New Chapters from The Molecular Probes® Handbook

Fluorescence Labeling and Detection Strategies in

Cellular BiologyDaniel W. Beacham, PhD

Senior Staff Scientist, Research and Development

Molecular Probes, Cellular Imaging and Analysis

Daniel.Beacham@lifetech.com

2

| Life Technologies Proprietary & Confidential |

The Handbook on Fluorescence Detection

3

Dyes, Indicators and Affinity LabelsFluorescent Dyes

Alexa Fluor® 488

• MW – ~250 –1800 Da

• Emission λ: ~400–850+ nm

• Ext. coeff.: 20,000–250,000 M-1cm-1

• Designed for use in live and/or fixed cell applications, some are fixable

• Antibody & streptavidin conjugates

• Ion & voltage indicators

• Organelle stains

• Protein labeling

• Cell tracing

• Enzyme substrates

300 400 500 600 700 800

Fluo-4 Calcium IndicatorAlexa Fluor® Dyes

4

• MW: ~25,000-30,000 Da

• Emission λ: ~450–650 nm

• Ext. coeff.: 5,000–150,000 M-1cm-1

• Designed for use in live cell applications, fixable

• Genetically encoded

• Protein expression

• Living biosensors

• Addressable organelle and cellular landmarking

• DIY tagging, FRET assay, trafficking tools

Fluorescent Proteins

Fluorescent QDot® Nanocrystals• Antibody & streptavidin

conjugates

• Cell tracing

• Vascular imaging

• Electron microscopy

• MW: Not applicable

• Emission λ: ~450–850 nm

• Ext. coeff.:140,000–8,000,000 M-1cm-1

• Primary use fixed cell applications, with limited live cell applications

• Photostable

Fluorescent Proteins & Nanocrystals

5

Overview of TechnologiesImaging ToolboxIndicator and Labeling Dyes

pHrodo™ pH sensor, Click-iT® chemistryBacMam Fluorescent Proteins

Targeted FPs and BiosensorsRhod-3 AM Calcium Indicator*CellEvent™ Caspase 3/7 Sensor*CellROX™ Deep Red ROS Sensor

HCS ToolBoxHCS CellMask™ StainsHCS Mitochondrial Health KitClick-iT® HCS ToolsMitotic Index Kit and HCS applications

6

Probing Compartmental pH with pHrodo™ Dyes

7

Phagocytic Index with pHrodo™ DyeDeltaVision™ Core

Side

Scat

ter

0200

800

600

1000

400

0 200 400 600 800 1000FSC-H

R1

Forward Scatter

100 101 102 103 104FL2 H

Counts

04

016

01

20

20

08

0Red Fluorescence

8

pHrodo™ Dextran for Endocytosis and Sorting

1.0

1.5

2.0

2.5

Vehicle Dynasore

IC50 Dynasore4.7 μM

9

Endocytosis with pHrodo™10K-dextran Initial signal seen in endosomes @ 10 mins

Endosome-GFP

Lysosome-GFP

…Trafficked into lysosomes @ 30 mins

Sorted away from Golgi and Peroxisomes

Peroxisome-GFP

Golgi-GFP

1)

2)

3)

10

CellLight™OVERLAY Endosome-GFP

AF-647-TfN pHrodo™ Dextran

pHrodo™ Dextran Multiplex

pHrodo™ Dextran conjugates are visible in endosomes and lysosomes

Transit through endosomes quickly*BacMam Endosome GFP*AF Transferrin Uptake

Emission is brighter in lysosomes than endosomes

Lysosomal pH is more acidic

11

Concentration dependence of the CFTRinh-172-induced inhibition of acidification in phagosomes and lysosomes.

Deriy et al. J. Biol. Chem. 2009;284:35926-35938

Disease-causing Mutations in the Cystic Fibrosis TransmembraneConductance Regulator Determine the Functional Responses of Alveolar Macrophages

pHrodo™ Dextran Ratiometry

0

500000

1000000

1500000

2000000

4 5 6 7 8

pH

12

pHrodo™ Labeled Antibody Internalization

In vivo fluorescence endoscopy

pHrodo SE Amine-Reactive dye for customized labeling

13

Click-iT® Chemistry for Modular Detection

14

What is Click-iT®?

• Small, bio-orthogonal and “interchangeable” functional groups

• Chemoselective ligation reaction

Two-step procedure:

1 ) Label molecule of interest metabolically, enzymatically or chemically

2) Versatile detection, compatible with any readout (fluorescence, absorbance), on any platform (blot, gel, flow cytometer, imaging, mass spectrometer)

Copper catalyzed click reaction between an alkyne and an azide

Nascent DNA Synthesis

15

Click-iT® Chemical BiologyAzide

Triazole

+Cu(I)

Room TempDNA

+

AF488

SubstrateSubstrateDNA RNA

ProteinPTM

AF488

Alkyne

16

Click-iT® EdU: Proliferation on Single Cell Level

BrdU Click-iT® EdU

17

Click chemistry EdU Protocol

TOTAL TIME70 minutes

Image

15 minutesWash 3X

15 minutesNuclear counterstain

10 minutesWash 2X

30 minutesClick-iT™ detection reaction

Incubate with EdU or BrdU, fix & permeabilize sample

With Click chemistry

• Measure proliferation in cells or tissue

• Time to complete:

18

Intact Cell Morphology with Click-iT ® EdUWhole Animal

Rat Ileum

Rat Brain

Cellular Label

Muntjac Cells

Salic, A. (2008) Proc Natl AcadSci USA 105:2415–2420

Muntjac skin fibroblasts with Click-iT® EdU Alexa Fluor® 647 (purple), immuno-detection of tubulin (blue), golgi (green) and peroxisomes (orange).

Nascent DNA Synthesis

19

Click-iT® EdU protocol BrdU protocol

Click-iT™ EdU Alexa Fluor® 594 2-color overlay

Green: Hoechst 33342. Red: EdU or BrdU detection with Alexa Fluor® 594 dye

Click-iT® EdU vs BrdU detection in tissue sections

20

Click-iT® EdU labeling birth-dates differentiated cells in mouse olfactory epithelium F. Chehrehasa et al. J NeurosciMethod (2009)

Click-iT® EdU Across Species (and Kingdoms)

Neural tube and otocystlabeling with Click-IT®EdU in chick embryos. M. Warren et al. Dev. Dynamics (2009)

Proliferating germ cells in C. elegans with Click-iT®EdU. M. Dorsett et al. Genetics (2009)

Image courtesy of Sarah Cheesman, University of Oregon

Methods (2009) 48:8-13Image courtesy of Julian P.S. Smith III, Winthrop Microscopy Facility, Winthrop University

E. colimarine flatworm zebrafish larva

Medicago sativa (alfalfa) suspension cultures labeled with Click-iT® EdU. Image courtesy of Ferhan Ayadin, Cellular Imaging Laboratory, Biological Research Center

S. pombeImage courtesy of Sarah A. Sabatinos, University of Southern California

21

Click-iT® Based Detection of Nascent RNA Synthesis

NIH 3T3 cells incubated with 1 mM EU for 1 hr followed by click reaction with Alexa Fluor® 647 azide (magenta), immuno-detection of tubulin with Alexa Fluor® 488 secondary (green), and Hoechst nuclear counterstain (blue).

EU Assay (5-ethynyl uridine)

22

Nascent RNA Imaging with Click-iT® EU

-EU+EU

HeLa cells +/- 1 mM EU for 1 hr followed by click reaction with Alexa Fluor® 488 azide (green), immuno-detection of tubulin with Alexa Fluor® 594 secondary (red), and Hoechst nuclear counterstain (blue)

23

Click-iT® Nascent Protein SynthesisA BB C

Untreated 150 µM cycloheximide

L-azido-homoalanine

(AHA)

H2N CO2H

35SMe

H2N CO2H

N3

35S-methionine

• Validated HCS kit that enables detection of pre-lethal effects of compounds on nascent protein synthesis

• Faster and safer alternative to radioactive methionine techniques

The Journal of Neuroscience, January 21, 2009. 29(3): 638-652U-2 OS cells

24

Primary human hepatocytesHoechst Click-IT® TUNEL Overlay

Control

10 µM A

ctinomycin

D

Click-iT® TUNEL Assay for DNA Damage

Stress/Ox DNA Nicking

25

Click-iT® Detection Assays…

Nascent DNA synthesis/Cell ProliferationClick-iT® EdU Cell Proliferation Assay

Nascent RNA SynthesisClick-iT® RNA Assay

Nascent Protein Synthesis Click-iT® Detection of Protein Synthesis & Post Translational

Modification

Detection of DNA Strand BreaksClick-iT® TUNEL Assay for Apoptosis

www.invitrogen.com/clickit

26

Click-iT® Precursors, Labels and Applications

Nascent protein, DNA, RNA synthesis Alexa Fluor® Dyes

Sugar PTM reagentsMannosylation Avidin/BiotinIsoprenylationFucosylation Oregon Green® 488Fatty AcylationO-Linked Glycoproteins: TAMRASialic Acid Modified Glycoproteins

Etc..TUNEL Assay

Azide/Alkyne Precursors Azide/Alkyne Labels:

27

Azide-modified molecule

Stable triazole conjugate

Fluorophore-, or hapten-DIBO

Copper-free Click-iT® Reagents for Live Cells

DIBO Alkynes react with incorporated azide macromolecules

28

Copper-Free Click Mannosylation in GFP Cells

Label:ManNAz Azido Mannose Label

Detect:Alexa Fluor® 647 DIBO alkyne

29

Targeted fluorescent proteins + BacMam

CellLight® Reagents

PremoTM Biosensors

BacMam Targets: Ion Channel, GPCR, Kinase etc

BacMam Gene Delivery

30

What is BacMam?• BacMam is the use of modified baculovirus on

mammalian cells in a way that is:– Convenient / easy-to-use

Just add to cells– Productive

High expression level– Safe

Non-toxic to cells and is BSL1No footprint; non-replicating, non-integrating

SF9cells Assay

transduceovernight

31

How Do I Use BacMam?

Actin RFP + Tubulin GFP

How Do I Use BacMam?

32

Human umbilical vein endothelial cellEndosome-GFP, Golgi-OFP

Human airway epithelial smooth muscleActin-RFP, Histone 2B-GFP

Human Aortic Smooth MuscleER RFP, Tubulin GFPDAPI

Adult Mouse Schwann CellGFP/Actin RFP

Human mesenchymalstem cellH2B RFP MAP4 GFP

Adipose Derived Stem CellsGFP Transduction Control

Primary Cells with No Toxicity

E18 Rat Hippocampal NeuronsRFP Tubulin

Human HepatocytesGFP H2B

Mouse CardiomyocyteGFP Actin, Hoerschst

33

BacMam CellLights® Targeted Fluorescent Proteins

34

Mouse Cardiomyocytes

Tubulin GFPActin GFP

35

Actin Stress Fiber Disruption

Tubulin GFPActin RFP

Cytochalasin D

Dissolves actin filamentsTubulin network intact

36

Hippocampal Neurons

GFP Control

MAP4 RFP

Actin RFP

Synaptophysin-RFPGFP + Mito RFP

37

Growth Cone Dynamics and Mito Traffic

GFP ControlMito RFP

38

Premo™ Cameleon Calcium Sensor

39

Premo™ Halide Sensor

0 10 20 30 40 50 60-20000

-15000

-10000

-5000

0

Seconds

ΔF

40

FUCCI Cell Cycle Sensor

Atsushi Miyawaki Lab, Riken Institute

GFP-Geminin+

RFP Cdt1=

FUCCI

41

BacMam Premo™ FUCCI Cell Cycle Sensor

Visualizing spatial and temporal aspects of cell cycle progression with fluorescent proteins via BacMam gene delivery technology

42

Premo™ LC3B Autophagy Sensor

LC3B

Glycine 120

Alanine 120 Alanine 120

Glycine 120

PE

Pro autophagic stimulus

Autophagosome formation

No Autophagosome formation

• Fusion between LC3B and GFP/RFP• Expressed by CMV-E1 on baculovirus

chromosome• Delivery via BacMam 2.0 technology• Transient expression in wide range of

mammalian cells• Live-cell indicator of autophagy

LC3B wt

LC3B mutant(control)

43

Premo™ LC3B Autophagy Sensor

Vehicle Chloroquine

Mu

tan

t

LC

3B

Vehicle Chloroquine

44

Autophagy in Primary Animal and Human CellsW

T LC

3B

LC

3BG

120A

Rat Hippocampal Human Aortic Smooth Muscle Cells

BacMam LC3B-GFP + CellLight® Golgi-RFP

45

Rhod-3 Calcium Imaging with CaV2.1

1 min

4X d

F/F

+5 mM K+

+15 mM K+

+30 mM K+

GFP MAP4Transmitted Light

BacMam CaV2.1 α + β +α2δ

Kd 570 nM

46

CellEvent™ Caspase 3/7 Green Apoptosis Sensor

DEVD DNA binding dye

Active Caspase-3/7 Enzyme

Non-fluorescentNo DNA binding DE

VD

DNA binding dye

Fluorogenic signal upon DNA binding

47

CellEvent™ Caspase 3/7 Imaging

C

A

D

BC

ON

Stau

rosp

orin

e

CellEvent™ Hoechst

AF 647 PhalloidinMitotracker RedHoerchst

AF 647 PhalloidinMitotracker Red

48

CellEvent™ Caspase 3/7 Time Lapse in HCSRed: TMRM Mitochondrial Vm Dye

Fades with apoptosis

Green: CellEvent™ Caspase 3/7Fluorogenic with apoptosis

Staurosporine Time Lapse

ImageXpress Micro®

49

CellROX™ Deep Red ROS Sensor1. Dihydrodichlorofluoresceins are traditionally used for ROS measurements.They have to

be added to serum free media and have higher backgrounds

2. CellROX™ Deep Red is a fluorogenic probe to measure oxidative stress in live cells.

3. Can be added to complete growth media.

4. CellROX™ Deep Red Reagent gives good S/N ratios and can be used with traditional fluorescence microscopy, high content screening, flow cytometry and flourescence plate reader assays.

5. CellROX™ Deep Red Reagent can be used in GFP-expressing cells and can also be multiplexed with other reagents to measure multiple parameters of cell health in the same cell.

50

CellROX™ Deep Red Staining

Plate cells(suspension cells, if using

flow cytometry, are grown in flasks)

Drug treatment

Add CellROX™ Deep Red reagent & Hoechst 33342

(incubate for 30 mins)

Wash cells 3X in PBS

Analyze

(Suspension cells are washed by centrifugation)Traditional Fluorescence Microscopy

High content screening

FluorescencePlate Reader

Flow Cytometry

0

2 00

4 00

6 00

8 00

10 00

12 00

14 00

16 00

18 00

Sig

nal I

nten

sity

(A

U)

Con tr ol Me na dion e

0

5 0

10 0

15 0

20 0

25 0

30 0

Mean

sig

nal i

nten

sity

C ont ro l Co nt rol + DP I LP S LP S + D PI

)

51

CellROX™ Deep Red ROS Sensor

Menadione

Live

cel

lsFi

xed

cells

Control

MenadioneControl

050

100150200250300350400450500

1

Mea

n flu

ores

cenc

e in

tens

ity

Live cells, control Live cells, menadioneFixed cells, control Fixed cells, menadione

U-2 OS Cells

52

Reactive Oxygen Imaging

Control 100 µM Menadione

Hoechst/CellROX™ Deep Red reagent

BPAE Cells

53

CellROX™ Deep Red reagent for detection of oxidative stress

Hoechst and CellROX™ Deep Red reagent added to live bovine pulmonary artery endothelial cells in complete media

Untreated 100 µM Menadione 100 µM Menadione + MnTBAP

Reduced fluoresceins, traditionally used for cellular ROS measurements, must be added to serum-free media and suffer from high background, photo-oxidation, photo-instability, etc.

Untreated

Untreated

Untreated

Untreated

500 nM Angiotensin-II

100 µM Menadione

50 µM Nefazodone

500 µg/mL LPS

RA

W c

ells

Hep

G2

cells

U-2

OS

cells

HA

SM c

ells

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

Time (sec)

Nor

mal

ized

Flu

ores

cenc

e In

tens

ity

CellROX™ Deep Red Reagent H2-DCFDA

54

CellROX™ Deep Red Continued

0

0.25 0.5 1 2 3 4

0

20

40

60

80

100

ROSCaspase 3/7

0.5 μM staurosporine (hours)

% P

ositi

ve C

ells

(+/-

SEM

)

CellROX™ Deep Red Reagent may also be used for flow cytometry, high content screening, and whole-well fluorescence plate reader assays

Untreated 4 hr staurosporine2 hr staurosporine

Y e sN oN oG F P c o m p a t ib ili ty

Y e sN oN oM u ltip lex ib il ity

Y e sN oN oF ix ab le

Y e sY e sN oA d d in C om p l et e m e d ia

Fa r R ed R O S S e n s orD ih yd r o e th i d i u m

D ic h lo r o fl uo re-sc e in d iac e ta te

Y e sN oN oG F P c o m p a t ib ili ty

Y e sN oN oM u ltip lex ib il ity

Y e sN oN oF ix ab le

Y e sY e sN oA d d in C om p l et e m e d ia

Fa r R ed R O S S e n s orD ih yd r o e th i d i u m

D ic h lo r o fl uo re-sc e in d iac e ta te

CellROX™ Deep Red, CellEvent Caspase 3/7 Green, andHoechst reagents added to live HeLa cells in complete media

55

The High Content Imaging Toolbox:Automated Microscopy and Analysis

HeLa - Valinomycin

-6 -4 -2 0 20

200

400

600 EC50 = 5.5 nM

Log Valinomycin (μM)

Mito

chon

dria

lM

embr

ane

Pote

ntia

l

HeLa - Valinomycin

-1 0 1 2 30

200

400

600

800

1000 EC50=25.5 μM

Log Valinomycin (μM)

Cel

l Mem

bran

ePe

rmea

bilit

y

56

HCS Segmentation ToolsStaining of Entire (Fixed) CellsHCS CellMask™ Blue stainHCS CellMask™ Green stain (new)HCS CellMask™ Orange stain (new)HCS CellMask™ Red stain (new)HCS CellMask™ Deep Red stain

Prominent Nuclear Staining in (Live or Fixed) CellsHCS NuclearMask™ Blue stain (new)HCS NuclearMask™ Red stain (“new”)HCS NuclearMask™ Deep Red stain

Fixable Plasma Membrane Staining in (Live) CellsCellMask™ Orange Plasma Membrane stainCellMask™ Deep Red Plasma Membrane stain

57

HCS Mitochondrial Health Kit0 nM 165 nM6 nM2 nM 120 μM40 μM13 μM4.4 μM

Hoechst 33342(Nuclear

Morphology)

MitoHealth stain(Mitochondrial

MembranePotential)

Image-IT® DEAD GreenTM Viability stain

(Cell MembranePermeability)

HeLa - Valinomycin

-6 -4 -2 0 20

200

400

600 EC50 = 5.5 nM

Log Valinomycin (μM)

Mito

chon

dria

lM

embr

ane

Pote

ntia

l

HeLa - Valinomycin

-1 0 1 2 30

200

400

600

800

1000 EC50=25.5 μM

Log Valinomycin (μM)

Cel

l Mem

bran

ePe

rmea

bilit

y

Image-IT® Dead:EC50= 25 uM

MitoHealth:EC50= 5.5 nM

58

ThiolTracker™ Violet for Glutathione Depletion Imaging

NuclearMask™Deep Red Stain

ThiolTracker™Violet Stain

Unt

reat

edTr

eate

d

Live HepG2 cells

59

Glutathione Depletion

• Effective marker for intracellular reduced glutathione (GSH)

• Easy to use and fixable• Deep red nuclear stain ideal for multiplexing• Much brighter than bimanes

Sensitive assay for signature cytotoxicity marker (GSH)

Control 125 µM BSO + 62.5 µM DEM 1 mM BSO + 0.5 mM DEM

Red: NuclearMask™ Deep RedGreen: ThiolTracker™ Violet

- 4 - 3 - 2 - 1 00

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

L o g ( m M ) , B S O + D E MThi

olTr

acke

r cy

topl

asm

ic in

tens

ity GSH depletion

BSO+DEM (Log mM)

Thio

lTra

cker

™Vi

olet

in

tens

ity

60

DNA Damage by phospho-H2AX Staining

H2AXp

Khanna, K.K. and Jackson, S.P. (2001) Nature Genet., 27, 247-254

ATR

DNA damage

(pH2AX antibody/

Alexa Fluor® 555 secondary)

Nuclear morphology

(Hoechst 33342)

Cell Membrane Permeability

(Image-iT® DEAD Green™

viability stain)

120 µM30 µMDMSO A549 Cells24h Valinomycin

61

HCS LIVE ⁄ DEAD® Green Kit Untreated Valinomycin (60 uM)

Nuclear morphology(HCS Nuclear Mask™Deep Red stain)Cell membrane permeability (Image-iT®DEAD Green viability stain)

Dye-based, two color cytotoxicity assay with fast and efficient workflowSensitive discrimination of live and dead cellsCompatible with fixation and permeabilization for combination with antibody-based assays

Fast and highly sensitive two-color cytotoxicity assay

62

Live Cell HCS with pHrodo™ e. coliImageXpress Micro®

63

HCS Mitotic Index Kit

Measures % of mitotic cells • Phospho-histone 3 is a known mitotic marker, commonly used in cancer research, toxicology and

cell cycle signaling• Phospho-histone H3 primary with Alexa Fluor® 488 secondary enables characterization and

quantitation of mitotic cells and compounds causing mitotic arrest• Kit contains DAPI or HCS NuclearMask™ Deep Red for total DNA content• Can be combined with other cell cycle markers to study cell cycle related and other physiological

pathways• Sensitive and accurate assay for mitotic arrest and toxicity

Proliferating, healthy cell

Phospho-histone H3 activation in cells arrested in mitosis

Cell damage

64

HCS Mitotic Index Kit

-6 -5 -4 -3 -2 -1 0 1 2 3 4 50

1 02 03 04 05 06 07 08 09 0

E C 5 0 = 3 6 n M

L o g (μ M ), V in c r is t in e

% M

itotic

cel

ls

500 n M n oco d azo le

Co nt rol

Excellent assay robustness with tight CV values and Z’ scores

-6 -5 -4 -3 -2 -1 0 1 2 3 40

1 02 03 04 05 06 07 08 09 0

E C 5 0 , p la t e 1 = 5 7 n ME C 5 0 , p la t e 2 = 6 2 n M

L o g (μ M ), N o c o d a z o le

% M

itotic

cel

ls Nocodazole

Vincristine

Log (μM) compound

% M

itotic

cel

ls%

Mito

tic c

ells

HCS NuclearMask™Deep Red Stain

phospho-H3 Ab / Alexa Fluor® 488 secondary

65

Breakthrough cell proliferation assay with Click-iT® EdU

• Non-radioactive

• Multiplex compatible but, strand separation requirement for anti-BrdU access, can affect:

• Ability for other antibodies to bind

• Morphology

• Ability for dyes that require dsDNA to bind efficiently, i.e., cell cycle dyes

• Non-radioactive

• No DNA denaturation required

• Simplified protocol

• Small molecule detection

• Multiplex compatible, including

• Other antibodies

• Dyes for cell cycle analysis

66

HCS Click-iT® EdU assay

Control

4.5 µM

aphidicolin

Hoechst 33342 EdU Overlay

00.00.10.20.30.40.50.60.70.80.91.01.1

A549U2OS

0.001 0.01 0.1 1 10 100

Hela

Aphidicolin Concentration (µM)

Res

pons

eDNA EdU Cyclin B1 Composite

Un

trea

ted

Noc

odaz

ole

cont

rol

M ch

loro

quin

e

μ50

M ro

siglit

azon

e

μ

125

0mM

acet

amin

ophe

nM

etop

osid

e

μ5

0

20

40

60

80

100 EdU (AF488 azide)BrdU (MoBU-1 AF594)

% P

ositi

ve C

ells

Cells fed with 10μM Edu

Drug treatment-23 hrs

Cells fed with 10μM BrdU

67

Nascent RNA synthesis block measured with HCS Click-iT® EU Assay

α-Amanitin 250nMα-Amanitin 30nMα-Amanitin 0nM

-6 -5 -4 -3 -20

20

40

60

80EC50=17pM

Actinomycin D (pM)

Circ

Spot

Ave

rage

Inte

nsity

NIH 3T3 cells (images)Treatment with α-Amanitin for 18 hr followed by 1 mM EU incubation for 1 hr, click rxn with Alexa Fluor® 488 azide (green) and nuclear counterstaining with Hoechst (blue).

HeLa cells (dose response)Treatment with actinomycin for 18 hr followed by 1 mM EU incubation for 1 hr and click rxnwith Alexa Fluor® 488 azide

68

Nascent Protein Imaging with HCS Click-iT® AHA

Validated HCS kit that enables detection of pre-lethal effects of compounds on nascent protein synthesis

Faster and safer alternative to radioactive methionine techniques

U2OS

-2 -1 0 1 2 3 40

50

100

150

200

250Plate 1Plate 2

EC50

Plate 123.67

Plate 220.02

Log10 Anisomycin (nM)

Mea

n Rin

g Ave

rage

Inte

nsity

U2OS

-3 -2 -1 0 1 2 30

100

200

300Plate 1Plate 2

EC50

Plate 13.673

Plate 23.323

Log10 Puromycin (µM)

Mea

n Rin

g Ave

rage

Inte

nsity

U2OS

-5 -4 -3 -2 -1 0 10

50

100

150

200

250Plate 1Plate 2

EC50

Plate 10.1088

Plate 20.07522

Log10 Cycloheximide (µM)

Mea

n Rin

g Ave

rage

Inte

nsity

A549

-5 -4 -3 -2 -1 0 10

100

200

300Plate 1Plate 2

EC50

Plate 10.7417

Plate 20.2914

Log10 Cycloheximide (µM)

Mea

n Rin

g Ave

rage

Inte

nsity

Drug Titrations Performed in Duplicate

69

Co n tr ol 5 µ M C a m pt o th ec in 4 0 µ M C am p t ot he c in

- 5 - 4 - 3 -2 - 1 0 1 2 3

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 00

EC 5 0 = 9. 5 μM

L o g ( μ M ) , C a m p to t h e c in

% M

axim

um o

f TU

NE

L st

ain

ing

- 4 - 3 - 2 - 1 0 1 2 320 0

30 0

40 0

50 0

60 0

E C 50 = 8 nM

L og ( nM) , No c oda z ol e

Nuc

lear

Inte

nsi

ty (

TUN

EL)

Apoptosis in HepG2 cellsApoptotic HeLa cells in the HCS Click-iT® TUNEL Assay

No Treatment

2 μM Staurosporine

70| Life Technologies Proprietary & Confidential | 8/16/2011

Apoptosis in HCS with CellEvent™ Caspase 3/7 Reagent

-2 -1 0 1 20

10

20

30

40

Log [caspase 3/7 inhibitor 1], μM

% c

aspa

se-3

/7 p

ositi

ve

CellEvent™ Green Fluorogenic Caspase 3/7 substrateNo Inhibitor 20 µM Inhibitor

H

G

F

E

D

C

B

A

242322212019181716151413121110987654321

H

G

F

E

D

C

B

A

242322212019181716151413121110987654321

Con

trol

s

Con

trol

s

Con

trol

s

Con

trol

s

High content screening for activation of caspase 3/7-4 -3 -2 -1 0

0

20

40

60

80

100

Log [staurosporine], μM

% P

ositi

ve C

ells

(+/-

SEM

)

EC50 = 0.03 µM

71

CellROX™ Deep Red ROS Sensor in Live and Fixed

Menadione

Live

cel

lsFi

xed

cells

Control

MenadioneControl

050

100150200250300350400450500

1

Mea

n flu

ores

cenc

e in

tens

ity

Live cells, control Live cells, menadioneFixed cells, control Fixed cells, menadione

U-2 OS Cells

72

HCS LipidTOX™ phospholipidosis and steatosis

Kits include:

LipidTOX™ Red phospholipid stain

LipidTOX™ Green neutral lipid stain

Propranolol

Cyclosporin A

Hoechst 33342

HepG2 cells, 10 μM amiodarone48 hours

*LipidTOX™ probes also available as “stand alone”reagents

73

LipidTOX™ neutral lipid stains for adipogenesis

Adipocytes differentiated from human mesenchymalstem cells, fixed, and labeled with LipidTOX™ Green or Deep Red neutral lipid stain and Hoechst 33342

Adipocytes differentiated from 3T3 L1 mouse fibroblasts, fixed, and labeled with LipidTOX™ Green neutral lipid stain, anti-FABP4 (red), and Hoechst 33342

74

HCS Immunodetection of LC3B

0

100

200

300

400

500Mean ring spot avg intensity

control50μM

Chloroquine

control50μM

Chloroquinecontrol50μM

Chloroquine

25μM M

G132

HeLa A549 HepG2

0.5ug/ml Anti-LC3B

| 8/16/2011

75

http://www.invitrogen.com/hcs

76

Web-based Accessories

www.invitrogen.com/spectraviewer

Spectra Viewer

www.invitrogen.com/cellstaintool

The Virtual Cell

Questions? techsupport@lifetech.com / 800.955.6288

FacebookMolecular Probes Handbook Club

10,000+ Members since Dec 2010https://www.facebook.com/ProbesHandbook

FacebookCell Imaging

25,000+ Members since Dec 2010

https://www.facebook.com/cellimaging

77

Cell Imaging App for iPhone, iPad and Android

http://itunes.apple.com/us/app/cell-imaging

Cell Features and Biology Reagents and Results Protocols and Info

78

Iain JohnsonMagnus Persmark

George HansonMike O’GradyMike Janes

Nicolas DolmanKevin ChambersScott Clarke**Michelle Yan**Bhaskar MandavilliKary Oakleaf

Summary

daniel.beacham@lifetech.com

• Molecular Probes is alive and well!!

• New dyes and new chemistry− Would like your input

• Fluorescent Proteins + BacMam − = EASY!− Would like your content

Questions?

Acknowledgement: