Post on 11-Mar-2020
transcript
Click-iT® EdU : proliferation
measurement
•Jean-Michel Cocchi•Invitrogen Cellular Analysis•jean-michel.cocchi@lifetech.com
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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
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What is “click” chemistry ?
Azide
Alkyne
Triazole
+Cu(I)
Room Temp
Alexa Fluor® 488 Dye
DNA DNA
+
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Br
Br
Br
Br
1970’s - (BrdU)
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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
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Ethynyl dU = dU alkyne Bromo dU
H
Nucleoside analogue structures
Click-analogue
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Chemistry
Click labeling doesn’t
require DNA
denaturation
Dye azide reacts with the
alkyne on the double stranded
DNA
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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
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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
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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
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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
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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
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Sensitivity of Click-iT™ EdU Alexa Fluor® 647
S-phase cells can be detected after only a 5 minute EdU pulse
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Detection of newly synthesized DNA in < 2 hours
EdUtubulinDAPI
HeLa cells pulsed with 10 µµµµM EdU: detected with Click-iT® Alexa Fluor® 488
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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
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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.
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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.
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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
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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
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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)
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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
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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
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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)
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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
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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
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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
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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
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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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