Transcript Document

Fluorescent Solutions in
Cell Biology
Recent Developments in Cellular
Imaging, Apoptosis, Viability and
Cell Proliferation
Dana Brozban
Technical Sales Specialist
Imaging and Microscopy
This Presentation
• Focus on Key Molecular Probes Products and Applications
• “The Illuminated Cell”
• Apoptosis Assays, Viability and Cell Proliferation
2
Nucleus
T3605
S34857
S7020
S11368
D1306
H3570
SYTO®
SYTO®
SYTO®
SYTO®
SYTO®
TO-PRO®-3 iodide
SYTOX® Blue
SYTOX® Green
SYTOX® Orange
DAPI
Hoechst 33342
9 dye
13 dye
16 dye
25 dye
59 dye
Nucleoli
S32703 SYTO® RNASelect™ green
fluorescent cell stain
Endoplasmic Reticulum
E12353
ER-Tracker™ Blue-White DPX
E34250
ER-Tracker Red™
E34251
ER-Tracker Green™
S34200
SelectFX™ Alexa Fluor® 488 Endoplasmic Reticulum Labeling Kit
B7449
Brefeldin A, BODIPY® 558/568 conjugate *isomer 1*
ER-Tracker™ Family:
Live Cell Endoplamic Reticulum Markers
E12353
ER-Tracker™ Blue-White DPX
E34251
ER-Tracker Green™
E34250
ER-Tracker Red™
Golgi
A21270
Cells)
anti–golgin-97 Ab (Primary Ab or fixed
N22651
NBD C6-ceramide complexed to BSA
B22650
BSA
BODIPY® FL C5-ceramide complexed to
B34400
BSA
BODIPY® TR C5-ceramide complexed to
Mitochondria
M7512
MitoTracker® Red CMXRos
M7514
MitoTracker® Green FM
M7510
MitoTracker® Orange CMTMRos
T3168
JC-1
A6403
anti–cytochrome oxidase subunit I
(from panel of Ox Phos Complex Primary Ab´s
directed against Mitochondrial Enzymes)
Lysosomes
L7528 LysoTracker® Red DND-99
L7526 LysoTracker® Green DND-26
L7545 LysoSensor™ Yellow/Blue DND160
Peroxisomes
S34201 SelectFX™ Alexa Fluor® 488 Peroxisome Labeling Kit
Cytoskeleton/Tubulin
T34075 TubulinTracker™ Green
A11126 anti–α-tubulin mouse monoclonal IgG
TubulinTracker™ Green
Live BPAE cells + Oregon Green Taxol
Live BPAE cells + TubulinTracker™ Green
Drastically improved tubulin labeling in living cells
Cytoskeleton/Actin
A12379
Alexa Fluor® 488
phalloidin
R415
rhodamine phalloidin
A12381
Alexa Fluor® 594
Cytosol
C2925
CellTracker™ Green CMFDA
C34552
CellTracker™ Red CMTPX
C3100MP Calcein, AM
New
Qtracker Family
Three color Qtracker Cell Labeling
Plasma Membrane
V22885 Vybrant® DiI cell-labeling solution
V22886 Vybrant® DiO cell-labeling solution
V22887 Vybrant® DiD cell-labeling solution
W11262 Alexa Fluor® 594 wheat germ agglutinin
F35355 FM® 1-43FX *fixable analog of FM® 1-43 membrane
Lipid Rafts
B13950 BODIPY® FL C5-ganglioside GM1
V34403 Vybrant® Alexa Fluor® 488 Lipid Raft Labeling
Kit
V34404 Vybrant® Alexa Fluor® 555 Lipid Raft Labeling
Kit
MitoSOXTM Red Oxidant Selectivity
Relative Fluorescence Units (580 nm)
70000
60000
50000
40000
30000
20000
10000
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MitoSOXTM Red - Highly Selective Superoxide Detection
In Mitochondria of Live Cells
MitoSOX™ Red mitochondrial superoxide indicato
Buffer for 10 min
5 mM MitoSOXTM Red
Xanthine oxidase
pre-oxidation of 5 mM MSR
10 mM FeTCPP for 10 min
5 mM MitoSOXTM Red
No accumulation
in mitochondria
MitoSOX Red oxidation prevention by
superoxide scavengers in live BPAE cells
Cell tracers: membrane probes
Classic Example: DiI
(pronounced “dye eye”)
• Diffuse along membranes
• Useful in living or fixed tissues
• Long retention time (weeks)
• Generally not well retained
upon fixation
• Workhorses for neural tracing
Cell tracers: dextrans and protein tracers
Classic examples:
fluoro-ruby and fluoro-emerald
10,000 MW dextrans labeled with either
rhodamine (ruby) or fluorescein (emerald)
• Must be loaded by injection
• Long retention time
• Some variants are fixable
• Group includes protein tracers such
as HRP and cholera toxin subunit B
Cell tracers: polar tracers
Classic examples: Lucifer Yellow and Sulforhodamine 101
• Similar to dextran tracers, but
are low molecular weight
• Must be loaded by injection or
patch pipette
• Some variants are fixable
• May cross gap junctions
• Also includes labeled hydrazides
and biocytin
Myelin Staining Protocol Comparisons
™
FluoroMyelin™ is More Efficient
FluoroMyelin Green™ and FluoroMyelin™ Red
Arbor vita of mouse cerebellum sagittal cryosection
labeled with FluoroMyelin™ Red, NeuroTrace™
Green Nissl Stain, and DAPI.
Mouse rostral mesencephalon cross-section labeled with
FluoroMyelin™ Green, NeuroTrace™ Red Nissl Stain,
and DAPI. This combination is sold as the new
BrainStain™ Imaging Kit.
Single-step dyes requiring only 20 minutes label time
BrainStain Kit
The BrainStain™ Imaging Kit:
–DAPI
–NeuroTrace™ Red Nissl Stain
–FluoroMyelin™ Green
One 20-minute label step…
…three-color staining for
general anatomical
visualization,
or use the dyes separately.
Mouse diencephalon cross-section
Three Labels in One Step
Navigation
• Viability and vitality reagents
– LIVE/DEAD® kits
• Apoptosis Reagents
–
–
–
–
–
Apoptosis “timeline”
Annexin V conjugates & kits
Mitochondrial reagents
Vybrant® FAM caspase assay kits
Metabolic and nuclear changes
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Viability & Vitality
Viability:
Cell membrane forms intact barrier
Cytosol
Vitality:
Cell mediates active processes
Cytosol
Nucleus
Nucleus
+ Carboxyfluorescein
+ Propidium
iodide
Nonviable (Dead)
Viable (Live)
Nonvital (Dead)
Vital (Live)
How it works
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Viability & Vitality: Individual Reagent Examples
Viability
Vitality
Permeant DNA
Dyes (Live & Dead)
Impermeant DNA
Dyes (Dead)
Cell Function Markers
•
•
•
•
•
•
•
•
Esterase
SYTO® 9 dye
SYTO® 13 dye
SYTO® 16 dye
SYTO® 25 dye
SYTO® 59 dye
•
•
•
SYTOX® Blue
SYTOX® Green
Propidium iodide
(PI)
7-AAD
TO-PRO®-3 dye
Ethidium
homodimers
•
•
CFDAs
Calcein AM
Oxidation
•
•
Dihydrorhodamine 123
C12-resazurin (Vybrant®
Cell Metabolic Assay)
Mix & match based on spectral overlap
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Vitality by Enzyme / Metabolic Function
Dead Cell
Live Cell
Calcein AM
Ethidium
Homodimer-1
Cytosol
Nucleus
AM cleavage
Laser
“It isn’t easy being green”
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Vitality: LIVE/DEAD® Viability / Cytotoxicity Kit
(L3224)
Calcein AM and Ethidium Homodimer-1
•
•
•
Rapid assay
Detects live and dead cells simultaneously
The most popular viability assay kit
Vitality = metabolic activity
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New: LIVE/DEAD® Cell Vitality Assay Kit (L34951)
C12 resazurin + SYTOX® Green dye
Metabolic activity and viability
488 & 633 nm excitation
C12 resazurin
alamarBlue
100
% fluorescence remaining in cells
Jurkat cells induced with 10 μM
camptothecin for 4 hours, then stained
with the LIVE/DEAD® Cell Vitality Assay Kit
SYTOX Green dye fluorescence
•
•
•
75
50
25
0
0
10
20
30
40
Time (minutes)
50
60
120
C12 resazurin fluorescence
Increased permeability / brighter signals than alamarBlue
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Viability: Kit Examples
LIVE/DEAD®
Sperm Viability Kit (L7011)
SYBR® 14 dye and propidium iodide
LIVE/DEAD® Reduced Biohazard
Cell Viability Kit #2 (L23101)
Healthy cells
Aged culture
LIVE/DEAD® Reduced Biohazard Cell Viability Kit
#2 Principal
Heat-killed cells
Viability = membrane integrity
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New: Cell Vitality
L34955 LIVE/DEAD® Fixable
Violet Dead Cell Stain Kit
•Cytoplasmic Labeling
•Fixable Assay
•Ideal for work with pathogens
•Also available in Red, Green, and Blue
S34857 SYTOX® Blue Dead
Cell Stain
•Nucleic Acid Labeling
•Excites with 405nm laser
•Quantitative, single-step dead cell
indicator
•Minimal Spectral Overlap
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New Package Sizes: CellTrace™ Kits
C34554 CellTrace™ CFSE Cell Proliferation Kit
C34555 CellTrace™ Oregon Green® SE
•
•
•
•
New packagings in easy to use
aliquots
Track cell divisions over multiple
generations
Existing product repackaged in
“experiment-size” aliquots for ease
of use and maximum shelf-life
488 nm excitation
Initial Intensity
7th doubling
Increased divisions = decreased fluorescence
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CyQUANT Cell Proliferation Assay
Quick and Easy No washes, extractions or long incubations
Convenient Samples can be frozen
Sensitive and Accurate Linear from 50 to 50,000 cells; specific for
total nucleic acids
No Radioactivity An excellent alternative to 3H-thymidine
incorporation assays
X
New CyQuant NF – No Freeze
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New: Cell Cycle Analysis
V35003 Vybrant® DyeCycleTM Violet Stain
G0/G1
•Identifies major phases of cell cycle
-G0/G1, S, G2/M
•Excellent tool for Violet laser
•Also Available:
•V35004 Vybrant® DyeCycleTM Green
•V35005 Vybrant® DyeCycleTM Orange
S
G2/M
DNA Content Analysis in LIVING Cells
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Viability & Vitality: LIVE/DEAD® Kits
Sku #
Kit Name
Live Stain
Dead Stain
Excitation
Fix?
L3224
LIVE/DEAD® Viability/
Cytotoxicity Kit
Calcein AM
Ethidium Homodimer1
488nm
No
L7010
LIVE/DEAD® CellMediated/Cytotoxicity Kit
DiOC18(3)
Propidium Iodide
488nm
No
L7013
LIVE/DEAD® Reduced Biohazard
Cell Viability Kit #1
SYTO® 10 dye
Reactive Red
488nm
Yes
L23101
LIVE/DEAD® Reduced Biohazard
Cell Viability Kit #2
Blue Dye
dim
Blue Dye
bright
UV
Yes
L23102
LIVE/DEAD® Reduced Biohazard
Cell Viability Kit #3
Red Dye
dim
Red Dye
bright
488nm
Yes
L23105
LIVE/DEAD® Reduced Biohazard
Cell Viability Kit #4
Green Dye
dim
Green Dye
bright
488nm
Yes
L34955
New
LIVE/DEAD® Fixable
Violet Dead Cell Stain Kit
Violet Dye
dim
Violet Dye
bright
405 nm
Yes
L7011
LIVE/DEAD® Sperm Viability Kit
SYBR® 14 dye
Propidium Iodide
488nm
No
LIVE/DEAD® lineup: pick your colors
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Apoptosis – Programmed Cell Death
Ca2+
Chemical or
γ Irradiation
Bcl-2
dATP
pCasp-9
Apoptosome
Cyto c
APAF-1
APAF-1
Cyto
pCasp-9
O2
Calpain
Activation
Ca2+
O2
NAD
Cyto c
ΔΨm
Energy
c
Effector Caspase-3
Caspase Caspase-6
Caspase-7
PARP
Mitochondria
Free Radicals
EndoG
AIF
Caspase-9
DNA Fragmentation
Apoptosis
One result … many avenues
DNA Damage
Necrosis
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Timeframes within Camptothecin / Jurkat Model*
Function
Reagent
Mitochondria
Activity Changes
Membrane Potential
Transition Pore
MitoTracker® Red dye
DiOC2(3), DiIC1(5), JC-1
MTP assay
PS Translocation
Annexin
Caspase Activity
Caspase substrates
Metabolic Activity
C12 resazurin
DNA Condensation
Hoechst
Membrane Integrity
PI, SYTOX® Green dye
DNA Fragmentation
TUNEL
Increase
Decrease
Partial Change
Relative Time
ASSUMED
* Jurkat cells induced with 10 µM camptothecin
Putting it into a framework
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Loss of Membrane Asymmetry: Annexin V
Ca2+
Ca2+
•
•
Chemical or
γ Irradiation
O2
Calpain
Activation
Bcl-2
Annexin V Conjugates dATP
– Alexa Fluor® 350 (346/442)
pCasp-9
Cyto
®c 488 (495/519)
–
Alexa
Fluor
APAF-1
Cyto c
ΔΨm
Apoptosome
APAF-1
Cyto(496/519)
c
– Fluorescein
pCasp-9
Effector Caspase-3
– Oregon Green®Caspase
488 (496/524)
Caspase-6
EndoG
Caspase-7
– Alexa Fluor® 568 (578/603)
Caspase-9
– Alexa
Fluor® 594 (590/617)
– Alexa Fluor® 647 (650/668)
DNA Fragmentation
– Allophycocyanin (650/660)
– Biotin
Vybrant Apoptosis Kits (annexin,
viability, vitality)
Apoptosis
PARP
O2
NAD
Energy
Mitochondria
Free Radicals
AIF
DNA Damage
Necrosis
Probes to detect plasma membrane changes
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Loss of Membrane Asymmetry: Annexin V
Jurkat cells stained with
Vybrant® Apoptosis Assay Kit #2
(V13241)
Untreated cells
Dead
Viable
Apoptotic
Induced with camptothecin
Dead
Viable
Apoptotic
Detection of phosphatidyl serine translocation
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Selection Guide for Vybrant® Apoptosis Assay Kits
Kit #
Cat #
Viable Cell Stain
Apoptotic Cell Stain
Dead Cell Stain
Excitation
Kit #1
V13240
None
Alexa Fluor® 488 dye Annexin V
SYTOX® Green
488nm
PI (Red)
488nm
Kit #2
V13241
None
Alexa Fluor® 488 dye Annexin V
Kit #3
V13242
None
FITC-Annexin V
PI (Red)
488nm
YO-PRO®-1 dye
PI (Red)
488nm
Kit #4
V13243
None
YO-PRO®-1 dye (Green)
Kit #5
V13244
Hoechst 33342 dim
Hoechst 33342 (Blue)-Bright
PI (Red)
UV & 488nm
Kit #7
V23201
Hoechst 33342 dim
YO-PRO®-1 dye
Hoechst 3342
YO-PRO®-1 dye
PI (Red)
UV & 488nm
Kit #8
V35112
None
R-Phycoerythrin-Annexin V
(Orange)
SYTOX® Green dye
488nm
Kit #9
V35113
None
Allophycocyanin-Annexin V
(Far Red)
SYTOX® Green dye
488nm &
633nm
Kit #10
V35114
C12-resauzurin (Red)
Allophycocyanin-Annexin V
(Far Red)
SYTOX® Green dye
488nm &
633nm
Kit #11
V35116
MitoTracker® Red dye
Alexa Fluor® 488 dye Annexin V
None
488nm
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MitoProbe™ Reagents: Detecting Mitochondrial Changes
Ca2+
Chemical or
γ Irradiation
dATP
pCasp-9
Apoptosome
Cyto c
APAF-1
APAF-1
Cyto
pCasp-9
O2
Calpain
Activation
Ca2+
Cyto c
Bcl-2
O2
NAD
ΔΨm
Energy
c
Effector Caspase-3
Caspase Caspase-6
Caspase-7
PARP
Mitochondria
Free Radicals
EndoG
AIF
Caspase-9
•
•
•
•
•
MitoProbe™ JC-1 Assay
(M34152)
DNAKit
Fragmentation
MitoProbe™ DiOC2(3) Assay Kit (M34150)
MitoProbe™ DiIC1(5) Assay Kit (M34151)
MitoProbe™ Transition Pore Assay Kit (M34153)
Vybrant® Apoptosis Assay
Kit #11 (V35116)
Apoptosis
DNA Damage
Necrosis
Mitochondria: canary in the coal mine
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MitoProbe™ JC-1 Assay Kit (M34152)
JC-1 red fluorescence
•
•
•
•
Mitochondrial membrane potential
New packaging of classic dye with robust protocol
488 nm excitation – uses all emission
VIP Product - Widely Used
Jurkat cells + 2 μM JC-1
+ 50 μM CCCP
Decrease of red
JC-1 signal in
apoptotic cells
JC-1 green fluorescence
Classic reagent: optimized for flow cytometry
46
MitoProbe™ DiOC2(3) Assay Kit (M34150)
•
•
•
•
Mitochondrial membrane potential
488 nm excitation – uses all emissions
Easier to use than JC-1; better population
resolution
Can be used with APC-Annexin V conjugates for
excellent multiparameter study of Apoptosis and
Vitality
DiOC2(3) on HeLa cells
+CCCP
Decrease of red and
green DiOC2(3) signals
in apoptotic cells
Easy reagent – clear result
47
MitoProbe™ DiIC1(5) Assay Kit (M34151)
•
•
Mitochondrial membrane potential
633 nm excitation – allows use of 488 nm
emissions for other reagents
DiIC1(5) on MRC5 cells
Events
Jurkat cells stained with 50 nM DiIC1(5): 633 nm
Excitation
+50 μM
CCCP
Untreated
Decrease of far red
DiIC1(5)
fluorescence
DiIC1(5) fluorescence
Detection of mitochondrial changes with a 633nm laser
48
MitoProbe® Transition Pore Assay Kit (M34153)
•
•
•
New assay - early mitochondrial membrane event
Direct detection of the mitochondrial permeability transition pore (MPTP)
488 nm excitation & green emission; use of orange/red channels for other reagents
D
A
B
Whole cell fluorescence
F
Calcein
Alone
Calcein fluorescence
E
C
Mitochondrial fluorescence
G
Calcein
+ Cobalt
Calcein fluorescence
Diminished fluorescence
H
BPAE cells +
Calcein
calcein AM
/
+
Cobalt
CoCl2
+ Ionomycin
Calcein fluorescence
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MitoProbe® Transition Pore Assay Kit (M34153)
BPAE cells labeled with calcein AM, MitoTracker® Red dye, and Hoechst 33342
A
B
D
C
Seeing the assay with your own eyes
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Vybrant® FAM Caspase Assay Kits
Ca2+
Chemical or
γ Irradiation
Bcl-2
dATP
pCasp-9
Apoptosome
Cyto c
APAF-1
APAF-1
Cyto
pCasp-9
O2
Calpain
Activation
Ca2+
ΔΨm
c
EndoG
•
DNA Fragmentation
•
Apoptosis
Energy
Mitochondria
•
Caspase-9
O2
NAD
Cyto c
Effector Caspase-3
Caspase Caspase-6
Caspase-7
PARP
Free Radicals
AIF ®
Vybrant
FAM Caspase-3 and –7 Assay Kit
(V35118)
Vybrant® FAM Caspase-8 DNA
Assay
Kit
Damage
(V35119)
Vybrant® FAM Polycaspases Assay Kit
(V35117)
Necrosis
Probes to detect caspase activation
51
Vybrant® FAM Caspase Assay Kits: FLICA Function
Detection of active caspases in apoptotic cells
52
Vybrant® FAM Caspase Assay Kits
Propidium Iodine Fluorescence
• Vybrant® FAM Polycaspases Assay Kit (V35117)
• Vybrant® FAM Caspase-8 Assay Kit (V35119)
• Vybrant® FAM Caspase-3 and –7 Assay Kit (V35118)
Dead Cells
PI Positive
Increase of green FAM-FLICA
Fluorescence in apoptotic cells
Live Cells
FAM Negative
Apoptotic Cells
FAM Positive
FAM Fluorescence
Reliable detection of active caspases
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Example of multiparameter analysis
• Easily combine studies of caspase activation with membrane asymmetry
FAM-polycaspase
Green Channel
Propidium Iodine
Red Channel
APC-Annexin V
Far Red Channel
FAM Polycaspase
fluorescence
Live cells, annexin V-positive, PI-positive
30 minutes
1 hour
2
hours
3
hours
APC annexin V fluorescence
•Note the shift in APC fluorescence (Green arrow) prior to shift in FAM fluorescence (Blue
arrows)
•Allows study of timing of apoptoic events (I.e. membrane asymmetry prior to caspase
activation)
Multiparameter: caspase activity & membrane assymetry
54
Detecting Metabolic and Nuclear Changes
Ca2+
Chemical or
γ Irradiation
O2
Calpain
Activation
Ca2+
Bcl-2
dATP
pCasp-9
Apoptosome
Cyto c
APAF-1
APAF-1
Cyto
pCasp-9
Cyto c
O2
NAD
ΔΨm
Energy
c
Effector Caspase-3
Caspase Caspase-6
Caspase-7
PARP
Mitochondria
Free Radicals
EndoG
AIF
Caspase-9
•
•
•
Vybrant® Apoptosis Assay
#10
DNAKit
Fragmentation
(V35114)
Vybrant® Apoptosis Assay Kit #5
(V13244)
APO-BrdU™ TUNEL Assay Kit
(A23210)
Apoptosis
DNA Damage
Necrosis
Metabolic & nuclear changes: the end game
55
Vybrant® Apoptosis Assay Kit #10 (V35114)
Allophycocyanin annexin V + C12 resazurin + SYTOX® Green dye
Membrane asymmetry, metabolic activity and membrane integrity
488 & 633 nm excitation
Jurkat cells treated as labeled: 488 nm & 633 nm
excitation
Live cells, annexin V-positive, SYTOX® Green-positive
A
B
C12-resazurin
4 hours
camptothecin
Reductase
resorufin
Necrotic
(peroxidetreated)
Resorufin fluorescence
•
•
•
C
D
APC annexin V
SYTOX® Green Dye
Reductase activity / metabolic health
56
Chromatin Condensation / DNA Fragmentation
Vybrant® Apoptosis Assay Kit #5 (V13244)
Hoechst 33342 / propidium iodide
APO-BrdUTM TUNEL Assay Kit (A23210)
Alexa Fluor® 488 anti-BrdU /
propidium iodide
Nuclear changes: the finale
57
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www.invitrogen.com/iProtocol
59
www.invitrogen.com/iPath
60
www.invitrogen.com/Antibodies
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Literature and brochures
Easy downloads from the web, or available on request.
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