Click-iT® EdU : proliferation measurement · “Click”chemistry is a concept first introduced by...

Click-iT® EdU : proliferation

measurement

•Jean-Michel Cocchi•Invitrogen Cellular Analysis•[email protected]

| Life Technologies Proprietary & Confidential | 2

Then came “Click ”………“Click” chemistry is a concept first introduced by K. Barry Sharpless in 2001 that describes a set of chemical reactions for use in chemical library synthesis. However the name stuck to one conjugation reaction first described by Tornoe, the modified Huisgen reaction, or the :

Kolb HC, Finn MG, Sharpless K.B.; (2001) Angewandte Chemie International Edition V40:2004-2021.

Tornoe CW, Christensen C, Meldal M; (2002) J Org Chem. May 3;67(9):3057-64.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)

•Highly efficient•Rapid•Bio-orthogonal•Components are inert to biomolecules•No side reactions

Highly sensitive detection of “click” labeled macromolecules

N NN

R''

R'

Macromolecule

DetectionProbe

Cu(I), RT, water, I hour

•Low background

•High Sensitivity

1,3 adduct


What is “click” chemistry ?

Azide

Alkyne

Triazole

+Cu(I)

Room Temp

Alexa Fluor® 488 Dye

DNA DNA

+


Br

Br

Br

Br

1970’s - (BrdU)


Acid or Acid or Acid or Acid or DNaseDNaseDNaseDNase

Br

Br

Br

Br

BrdU - is inaccessible to the BrdU antibody

Requires DNA denaturation for strand separation

Cell cycle dye requires the DNA to be double-stranded

Difficult balancing act:

Numerous protocols: acid, heat, or nuclease for DNA denaturation


Ethynyl dU = dU alkyne Bromo dU

H

Nucleoside analogue structures

Click-analogue


Chemistry

Click labeling doesn’t

require DNA

denaturation

Dye azide reacts with the

alkyne on the double stranded

DNA


Click-iT™ 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-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

15 minutesNuclear counterstain

15 minutesWash 3X

15 minutesWash 3X

15 minutesWash 3X

60 minutesBlock

1-16 hoursAnti-BrdU incubation

120 minutesSecondary antibody incubation

TOTAL TIME 6-21 hours

Image

15 minutesWash 3X

12 minutesNeutralize

40 minutesHCl Denaturation

15 minutesWash 3X

Click-iT® EdU vs. BrdU Workflow


BrdU-incorporated cells

EdU-incorporated cells

Anti-BrdU Alexa Fluor® 488

Click-iT™ EdU Alexa Fluor® 488

Click-iT™ Proliferation - BrdU vs. Click-iT™ EdU

Without DNA denaturation With DNA denaturation


Cell Cycle vs Click-iT™ EdU For Quantitating S-Phase

Channels (Red C-A-SytoxRed Cycle Red C-A)0 50 100 150 200 250

Nu

mb

er

020

040

060

0

Dip G1: 49.02 % at 53.17Dip G2: 11.93 % at 104.67Dip S: 39.05 %G2/G1: 1.97%CV: 5.20

Modfit

CellCycle 633-redChannels (Red C-A-SytoxRed Cycle Red C-A)0 50 100 150 200 250

Nu

mb

er

020

040

060

0

Dip G1: 49.02 % at 53.17Dip G2: 11.93 % at 104.67Dip S: 39.05 %G2/G1: 1.97%CV: 5.20

Modfit

CellCycle 633-red

Dyes for Cell Cycle Analysis

Proliferating cells are simply detected

Click-iT™ EdU

In this example, both methods calculate 39% of the total cell population are proliferating cells

Proliferating cells are mathematically

calculated using the signal from the

G0/G1 and G2/M populations

G2/M

G0/G1


Cell Cycle vs. Click -iT™ EdU Cell Proliferation

Dyes for Cell Cycle Analysis

Proliferating cells are

mathematically calculated

Click-iT™ EdU

• In this example, the Click-iT™ EdU measures only proliferating cells.

• With the cell cycle dyes proliferating cells are calculated relative to signals from 1N, 2N and 4N cells; due to the complexity, accuracy can decrease

Channels (Red C-A-660/20 cycle Red C-A)0 50 100 150 200 250

Nu

mb

er

020

040

060

080

0

CellCycle 633-redChannels (Red C-A-660/20 cycle Red C-A)0 50 100 150 200 250

Nu

mb

er

020

040

060

080

0

CellCycle 633-red

CellCycle 633-redCellCycle 633-red

Proliferating cells are simply detectedin the complex population


33% 50%

16%

H929 myeloma cell line Fix, Click & Perm, then stain CD138-rPE & CD38-FITC

17%

82%

AlexaFluor®647 azide

AlexaFluor®647 azide

Click-iT™ EdU – See a specific cell population


Sensitivity of Click-iT™ EdU Alexa Fluor® 647

S-phase cells can be detected after only a 5 minute EdU pulse


Detection of newly synthesized DNA in < 2 hours

EdUtubulinDAPI

HeLa cells pulsed with 10 µµµµM EdU: detected with Click-iT® Alexa Fluor® 488


Click-iT™ In-vivo100-200 µg EdU injected intraperitoneally and 3 days later, organs were harvested, fixed, embedded in paraffin and sectioned. Sections were then de-waxed and EdU was detected with a red-fluorescent-azide with a 5 minute click reaction. Then, the section was stained with the blue fluorescent dsDNA stain, Hoechst 33342.

Image courtesy of Adrian Salic, Harvard University

Mouse Intestine Mouse Brain


Click-iT® EdU in Adult Brain Tissue.

Adult Mouse brain section, an organ whose cells almost never divide.

A sole EdU-labeled cell can be easily identified on this brain section.

Courtesy of Adrian Salic, Harvard.

Research published in

Feb 2008, PNAS 105(7) 2415-2420.


Click-iT EdU on Cultured Cells.

Click-iT EdU

Hoechst

U2OS cells. Pulsed for 30 mins with EdU then fixed. 15 minute reaction at room temp with Alexa Fluor 647-azide, rinsed then counterstained with Hoechst 33258.


Click-iT™ S-Phase Detection by Multiplexed HCS Imaging

Click-iT™ EdU Alexa Fluor® 594 Cyclin B1 Alexa Fluor® 488 Composite Image

Nocodazole(170 nM)

G2/M Block, 24 hr

Control


Anti-GFP with Click-iT™ EdU

• Organelle Lights™ NE-GFP (O36213) detected with anti-GFP rabbit serum antibody (A6455), followed by Alexa Fluor® 488 goat anti-rabbit antibody (A11008)

• Click-iT™ EdU Alexa Fluor® 594 Imaging Kit (A10209)

• MitoTracker® Deep Red FM


Click-iT ®-based detection of RNA synthesis in U-2 OS cells

-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)


Inhibition of RNA synthesis measured with the Clic k-iT®

EU Assay (5-Ethynyluridine) in NIH 3T3 and HeLa cell s

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

-6 -5 -4 -3 -20

20

40

60

80

EC50=17pM

Actinomycin D (pM)

Circ

Spo

t A

vera

ge In

tens

ity

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


Click-iT® AHA Alexa Fluor® 488 Protein Synthesis

� 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

Log 10 Anisomycin (nM)

Mea

n R

ing

Ave

rage

Inte

nsity

U2OS

-3 -2 -1 0 1 2 30

100

200

300Plate 1Plate 2

EC50

Plate 13.673

Plate 23.323

Log 10 Puromycin (µM)

Mea

n R

ing

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

Log 10 Cycloheximide (µM)

Mea

n R

ing

Ave

rage

Inte

nsity

A549

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

100

200

300Plate 1Plate 2

EC50

Plate 10.7417

Plate 20.2914

Log 10 Cycloheximide (µM)

Mea

n R

ing

Ave

rage

Inte

nsity

Drug Titrations Performed in Duplicate


Labeling newly synthesized proteins with AHA : Replacing 35S-Met with Click-iT ®

H2N CO2H

35S

Me

H2N CO2H

N335S-Met Azidohomoalanine

(AHA)

Primary neurons from mouse embryoniccortex grown for 4 weeks in vitro (35S-Met developed for 18 hours on film)

15’ 30’ 60’ 90’ cntrl 15’ 30’ 60’ 90’

AHA 35S-Met

HeLa cells cultured with AHA and ManKyne

Sialic acid-containing glycoproteins (green)

Hoechst (blue)

newly synthesized proteins (red)

Newly synthesized glycoproteins (yellow)


OAcOAcO

OAc

NH

O

OAc

N3

Ac4GlcNAz

Intracellular

O-GlcNAc

N3

Feed cells 1–3 days

Alkyne detectionprobe

+

Metabolic labeling with azido sugars combined with click chemistry


TUNEL (TdT mediated d UTP Nick End Labeling)

‘The intensity of labeling detected by BrdUTPimmunochemistry is nearly four times that obtained using biotin-conjugated dUTP, twice that using digoxygenin-conjugated dUTP, and over eight times that using direct labeling with fluorochrome (fluorescein or BODIPY)-conjugated deoxynucleotides The greater labeling with BrdUTP may reflect more efficient incorporation of this nucleotide by terminal transferase because of its smaller size than nucleotides with bulky fluorochrome conjugates.’Li and Darzynkiewicz (1995).Cell Proliferation; 28:5 71-579.

0

2

4

6

8

10

12

14

16

18

20

dUTP EdUTP BrdUTP fluorescein-dUTP

BODIPY®FL-dUTP

biotin-dUTP

num

ber

of n

ucle

otid

es a

dded

per

min


No Treatment 2 µµµµM Staurosporine

Detection of apoptotic cells using the Click-iT ®

TUNEL Alexa Fluor ® Imaging Assay

•0.5 uM staurosporine•Click-iT® TUNEL Alexa Fluor® 594•Anti-cleaved caspase 3/Alexa Fluor® 488 GAR•Hoechst 33342

•0.5 uM staurosporine•Click-iT® TUNEL Alexa Fluor® 647•Anti-cleaved caspase 3/Alexa Fluor® 488 GAR•Anti-tubulin/ AlexaFluor® 555 GAR•Hoechst 33342

HeLa cells


The Click-iT ® 2-step labeling and detection approach

•Metabolic•Enzymatic•Chemical

Phosphoproteins

Glycoproteins

Total proteins

DNA/RNA

Lipids

Incorporate azide/alkyne tag into macromolecule of interest

•1D-2D gel imaging

•Western blotting

•Fluorescence microscopy

•Flow cytometry

•Molecular enrichment

•In situ hybridization

•Fluorescent•Biotinylated•Affinity tags

Click-iT label macromolecule with desired azide/alkyne probes


A growing toolbox of labeling compounds for proteins and protein PTMs

AHA

H2N CO2H

35S

Me

H2N CO2H

N3

HPG

Nacent Proteins (35S-replacement)•Azidohomoalanine (AHA)•Homopropargylglycine (HPG)

Ac4Fucose alkyne

OOAc

AcOOAc

NHO

OAc

N3

Ac4GalNAz

OAcOAcO

OAc

NH

O

OAc

N3

Ac4GlcNAzAc4ManNAz

OAcOAcO

OAcHN

O

OAc

N3

Glycoproteins•Sialic acids•O-linked surface/secreted•O-GlcNAc modified•Fucosylated

Isoprenylation•Farnesylation•Geranylgeranylation Farnesylation analog Geranylgeranylation analog

Fatty Acid Modification•Palmitoylation•Myristoylation Myristoylation analog Palmitoylation analog


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Click-iT® EdU : proliferation measurement · “Click”chemistry is a concept first introduced by...

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