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Fluorescent Solutions in
Cell Biology

Recent Developments in Cellular
Imaging, Apoptosis, Viability and
Cell Proliferation

Dana Brozban

Technical Sales Specialist

Imaging and Microscopy

2

This Presentation


Focus on Key Molecular Probes Products and Applications



“The Illuminated Cell”



Apoptosis Assays, Viability and Cell Proliferation


Nucleus

T3605

TO
-
PRO®
-
3 iodide

S
34857


SYTOX® Blue

S7020

SYTOX® Green

S11368

SYTOX® Orange

D1306

DAPI

H3570

Hoechst 33342


SYTO® 9 dye

SYTO® 13 dye

SYTO® 16 dye

SYTO® 25 dye

SYTO® 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

anti

golgin
-
97 Ab (Primary Ab or fixed
Cells)

N22651

NBD C
6
-
ceramide complexed to BSA

B22650

BODIPY® FL C
5
-
ceramide complexed to
BSA

B34400

BODIPY® TR C
5
-
ceramide complexed to
BSA

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 DND
-
160

Peroxisomes

S
34201

SelectFX™ Alexa Fluor®
488
Peroxisome Labeling Kit

Cytoskeleton/Tubulin

T34075

TubulinTracker™


Green

A11126

anti

α
-
tubulin mouse monoclonal IgG

TubulinTracker


Green

Drastically improved tubulin labeling in living cells

Live BPAE cells + Oregon Green Taxol


Live BPAE cells + TubulinTracker


Green

Cytoskeleton/Actin

A12379

Alexa Fluor® 488
phalloidin

R415

rhodamine phalloidin

A12381

Alexa Fluor® 594
phalloidin

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
stain*

Lipid Rafts

B13950

BODIPY® FL C
5
-
ganglioside GM1

V34403

Vybrant® Alexa Fluor® 488 Lipid Raft Labeling
Kit

V34404

Vybrant® Alexa Fluor® 555 Lipid Raft Labeling
Kit

V34405

Vybrant® Alexa Fluor® 594 Lipid Raft Labeling
Kit

MitoSOX
TM

Red Oxidant Selectivity

0
10000
20000
30000
40000
50000
60000
70000
Superoxide
Dye only
Superoxide + SOD
Superoxide + FeTCPP
HRP + H2O2
H2O2
DEANO
Peroxynitrite
Relative Fluorescence Units (580 nm)
MitoSOX
TM

Red
-

Highly Selective Superoxide Detection

In Mitochondria of Live Cells

MitoSOX Red oxidation prevention by

superoxide scavengers in live BPAE cells

Buffer for 10 min

5 mM
MitoSOX
TM

Red

10 mM FeTCPP for 10 min

5 mM MitoSOX
TM

Red

Xanthine oxidase

pre
-
oxidation of 5 mM MSR

No accumulation

in mitochondria

MitoSOX™ Red mitochondrial superoxide indicator


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

Mouse diencephalon cross
-
section

Three Labels in One Step

One 20
-
minute label step


…three
-
color staining for

general anatomical
visualization,

or use the dyes separately.


28

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

29

Viability:

Cell membrane forms intact barrier

Vitality:

Cell mediates active processes

Viability & Vitality

Cytosol

Nucleus

Cytosol

Nucleus

Viable (Live)

Vital (Live)

Nonviable (Dead)

Nonvital (Dead)

+ Carboxy
-

fluorescein

+ Propidium
iodide

How it works

30


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

Viability & Vitality: Individual Reagent Examples

Esterase


CFDAs


Calcein AM

Oxidation


Dihydrorhodamine
123


C
12
-
resazurin (Vybrant
®

Cell Metabolic Assay)


Permeant DNA
Dyes (Live & Dead)

Impermeant DNA
Dyes (Dead)

Cell Function Markers


Viability

Vitality

Mix & match based on spectral overlap

31

Vitality by Enzyme / Metabolic Function

Laser

Cytosol

Live Cell

Dead Cell

Calcein AM

Ethidium

Homodimer
-
1

Nucleus

AM cleavage

“It isn’t easy being green”

32

Calcein AM and Ethidium Homodimer
-
1

Vitality: LIVE/DEAD
®

Viability / Cytotoxicity Kit
(L3224)

Vitality = metabolic activity


Rapid assay


Detects live and dead cells simultaneously


The most popular viability assay kit

33

New: LIVE/DEAD
®

Cell Vitality Assay Kit (L34951)

C
12

resazurin fluorescence

SYTOX

Green dye fluorescence


C
12

resazurin + SYTOX
®

Green dye


Metabolic activity and viability


488 & 633 nm excitation

C
12

resazurin

alamarBlue

0

10

20

30

40

50

60

120

Time (minutes)

% fluorescence remaining in cells

100

75

50

25

0

Jurkat cells induced with 10
μM

camptothecin for 4 hours, then stained
with the LIVE/DEAD
®

Cell Vitality Assay Kit

Increased permeability / brighter signals than alamarBlue

34

Viability: Kit Examples

LIVE/DEAD
®

Reduced Biohazard
Cell Viability Kit #
2
(L
23101
)

Viability = membrane integrity

LIVE/DEAD
®

Reduced Biohazard Cell Viability Kit
#2 Principal

LIVE/DEAD
®

Sperm Viability Kit (L7011)

SYBR
®

14 dye and propidium iodide

Healthy cells

Aged culture

Heat
-
killed cells

35

New
: Cell Vitality

L
34955
LIVE/DEAD® Fixable

Violet Dead Cell Stain Kit


S34857 SYTOX® Blue Dead

Cell Stain


Cytoplasmic Labeling


Fixable Assay


Ideal for work with pathogens


Also available in Red, Green, and Blue


Nucleic Acid Labeling


Excites with 405nm laser


Quantitative, single
-
step dead cell

indicator


Minimal Spectral Overlap

36

New Package Sizes: CellTrace


Kits

C
34554
CellTrace™ CFSE Cell Proliferation Kit

C
34555
CellTrace™ Oregon Green
®

SE

Initial Intensity

7
th

doubling


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

Increased divisions = decreased fluorescence

37

Convenient

CyQUANT Cell Proliferation Assay

No washes, extractions or long incubations

Samples can be frozen

Linear from
50
to
50
,
000
cells; specific for
total nucleic acids

An excellent alternative to
3
H
-
thymidine
incorporation assays


Quick and Easy

Sensitive and Accurate

No Radioactivity

X

New CyQuant NF


No Freeze

38

New
: Cell Cycle Analysis

G
0
/G
1

G
2
/M

S



Identifies major phases of cell cycle

-
G
0
/G
1
, S, G
2
/M



Excellent tool for Violet laser



Also Available:


V35004

Vybrant
®

DyeCycle
TM

Green


V35005

Vybrant
®

DyeCycle
TM

Orange

V
35003

Vybrant
®

DyeCycle
TM

Violet Stain

DNA Content Analysis in LIVING Cells

39

Viability & Vitality: LIVE/DEAD
®

Kits

Sku #

Kit Name

Live Stain

Dead Stain

Excitation

Fix?

L3224

LIVE/DEAD
®

Viability/
Cytotoxicity Kit


Calcein AM

Ethidium Homodimer
-
1

488nm

No

L7010

LIVE/DEAD
®

Cell
-
Mediated/Cytotoxicity Kit


DiOC
18
(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

40

Mitochondria

dATP

Calpain

Activation

Ca
2
+

Chemical or

γ Irradiation

Energy

Free Radicals

NAD

PARP

O
2

O
2

Bcl
-
2

Cyto c

Cyto c

Cyto c

APAF
-
1

APAF
-
1

pCasp
-
9

pCasp
-
9

Caspase
-
9

Effector

Caspase

Ca
2+


ΔΨ
m

AIF

EndoG

DNA Fragmentation

DNA Damage

Apoptosis

Necrosis

Caspase
-
3

Caspase
-
6

Caspase
-
7

Apoptosome

Apoptosis


Programmed Cell Death

One result … many avenues

41

Timeframes within Camptothecin / Jurkat Model*

Function

Reagent

Relative Time

PS Translocation

Annexin

Membrane Integrity

PI, SYTOX
®

Green dye

Mitochondria


Activity Changes


Membrane Potential


Transition Pore


MitoTracker
®

Red dye

DiOC
2
(3), DiIC
1
(5), JC
-
1

MTP assay

Caspase Activity

Caspase substrates

Increase

Decrease

Partial Change

Metabolic Activity

C
12

resazurin

* Jurkat cells induced with 10
µM camptothecin

Putting it into a framework

DNA Condensation

Hoechst

DNA Fragmentation

TUNEL

ASSUMED

42

Loss of Membrane Asymmetry: Annexin V

Mitochondria

dATP

Calpain

Activation

Ca
2+

Chemical or

γ Irradiation

Energy

Free Radicals

NAD

PARP

O
2

O
2

Bcl
-
2

Cyto c

Cyto c

Cyto c

APAF
-
1

APAF
-
1

pCasp
-
9

pCasp
-
9

Caspase
-
9

Effector

Caspase

Ca
2
+


ΔΨ
m

AIF

EndoG

DNA Fragmentation

DNA Damage

Apoptosis

Necrosis

Caspase
-
3

Caspase
-
6

Caspase
-
7

Apoptosome

Probes to detect plasma membrane changes


Annexin V Conjugates


Alexa Fluor
®

350
(
346
/
442
)


Alexa Fluor
®

488
(
495
/
519
)


Fluorescein (
496
/
519
)


Oregon Green
®

488
(
496
/
524
)


Alexa Fluor
®

568
(
578
/
603
)


Alexa Fluor
®

594
(
590
/
617
)


Alexa Fluor
®

647
(
650
/
668
)


Allophycocyanin (
650
/
660
)


Biotin


Vybrant Apoptosis Kits (annexin,
viability, vitality)

43

Loss of Membrane Asymmetry: Annexin V

Untreated cells

Jurkat cells stained with
Vybrant
®
Apoptosis Assay Kit #2
(V13241)

Induced with camptothecin

Apoptotic

Dead

Viable

Apoptotic

Dead

Viable

Detection of phosphatidyl serine translocation

44

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

Kit #2

V13241

None

Alexa Fluor
®

488 dye
-

Annexin V

PI (Red)

488nm

Kit #3

V13242

None

FITC
-
Annexin V

PI (Red)

488nm

Kit #4

V13243

None

YO
-
PRO
®
-
1 dye (Green)

YO
-
PRO
®
-
1 dye

PI (Red)

488nm

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

C
12
-
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

45

MitoProbe
™ Reagents: Detecting Mitochondrial Changes

Mitochondria

dATP

Calpain

Activation

Ca
2
+

Chemical or

γ Irradiation

Energy

Free Radicals

NAD

PARP

O
2

O
2

Bcl
-
2

Cyto c

Cyto c

Cyto c

APAF
-
1

APAF
-
1

pCasp
-
9

pCasp
-
9

Caspase
-
9

Effector

Caspase

Ca
2
+


ΔΨ
m

AIF

EndoG

DNA Fragmentation

DNA Damage

Apoptosis

Necrosis

Caspase
-
3

Caspase
-
6

Caspase
-
7

Apoptosome


MitoProbe


JC
-
1 Assay Kit (M34152)


MitoProbe


DiOC
2
(3) Assay Kit (M34150)


MitoProbe


DiIC
1
(5) Assay Kit (M34151)


MitoProbe


Transition Pore Assay Kit (M34153)


Vybrant
®

Apoptosis Assay Kit #11 (V35116)

Mitochondria: canary in the coal mine

46

JC
-
1 green fluorescence

JC
-
1 red fluorescence

+ 50 μM CCCP

Jurkat cells +
2
μM JC
-
1



Mitochondrial membrane potential


New packaging of classic dye with robust protocol


488
nm excitation


uses all emission


VIP Product

-

Widely Used

MitoProbe


JC
-
1
Assay Kit (M
34152
)

Decrease of red
JC
-
1
signal in
apoptotic cells

Classic reagent: optimized for flow cytometry

47

MitoProbe


DiOC
2
(
3
) Assay Kit (M
34150
)


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


Decrease of red and
green DiOC
2
(3) signals
in apoptotic cells

+CCCP

Easy reagent


clear result

DiOC
2
(
3
) on HeLa cells

48

MitoProbe


DiIC
1
(
5
) Assay Kit (M
34151
)

Events

DiIC
1
(5) fluorescence

+50 μM
CCCP

Untreated

Jurkat cells stained with
50
nM DiIC
1
(
5
):
633
nm

Excitation




Mitochondrial membrane potential


633
nm excitation


allows use of
488
nm
emissions for other reagents


Detection of mitochondrial changes with a
633
nm laser

Decrease of far red
DiIC
1
(5)
fluorescence

DiIC
1
(5) on MRC5 cells

49

MitoProbe
®

Transition Pore Assay Kit (M
34153
)

BPAE cells +
calcein AM /
CoCl
2

Calcein

+ Cobalt

Calcein

+ Cobalt

+ Ionomycin

Calcein fluorescence

F

G

H

Calcein

Alone

Calcein fluorescence

Calcein fluorescence

Whole cell fluorescence

Mitochondrial fluorescence

Diminished fluorescence


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

A

B

C

D

E

50

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

51

Vybrant
®

FAM Caspase Assay Kits

Mitochondria

dATP

Calpain

Activation

Ca
2
+

Chemical or

γ Irradiation

Energy

Free Radicals

NAD

PARP

O
2

O
2

Bcl
-
2

Cyto c

Cyto c

Cyto c

APAF
-
1

APAF
-
1

pCasp
-
9

pCasp
-
9

Caspase
-
9

Effector

Caspase

Ca
2
+


ΔΨ
m

AIF

EndoG

DNA Fragmentation

DNA Damage

Apoptosis

Necrosis

Caspase
-
3

Caspase
-
6

Caspase
-
7

Apoptosome

Probes to detect caspase activation


Vybrant
®

FAM Caspase
-
3
and

7
Assay Kit
(V
35118
)


Vybrant
®

FAM Caspase
-
8
Assay Kit
(V
35119
)


Vybrant
®

FAM Polycaspases Assay Kit
(V
35117
)

52

Vybrant
®

FAM Caspase Assay Kits: FLICA Function

Detection of active caspases in apoptotic cells

53

Vybrant
®

FAM Caspase Assay Kits


Vybrant
®

FAM Polycaspases Assay Kit (V35117)


Vybrant
®

FAM Caspase
-
8 Assay Kit (V35119)


Vybrant
®

FAM Caspase
-
3 and

7 Assay Kit (V35118)



FAM Fluorescence

Propidium Iodine Fluorescence

Dead Cells

PI Positive

Apoptotic Cells

FAM Positive

Live Cells

FAM Negative

Increase of green FAM
-
FLICA

Fluorescence in apoptotic cells

Reliable detection of active caspases

54

Example of multiparameter analysis

APC annexin V fluorescence

30
minutes

1
hour

2
hours

3
hours

FAM Polycaspase

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)

Live cells
,
annexin V
-
positive
,
PI
-
positive

Multiparameter: caspase activity & membrane assymetry


Easily combine studies of caspase activation with membrane asymmetry




FAM
-
polycaspase


Green Channel




Propidium Iodine Red Channel





APC
-
Annexin V


Far Red Channel

55

Detecting Metabolic and Nuclear Changes

Mitochondria

dATP

Calpain

Activation

Ca
2
+

Chemical or

γ Irradiation

Energy

Free Radicals

NAD

PARP

O
2

O
2

Bcl
-
2

Cyto c

Cyto c

Cyto c

APAF
-
1

APAF
-
1

pCasp
-
9

pCasp
-
9

Caspase
-
9

Effector

Caspase

Ca
2
+


ΔΨ
m

AIF

EndoG

DNA Fragmentation

DNA Damage

Apoptosis

Necrosis

Caspase
-
3

Caspase
-
6

Caspase
-
7

Apoptosome


Vybrant
®

Apoptosis Assay Kit #
10
(V
35114
)


Vybrant
®

Apoptosis Assay Kit #
5
(V
13244
)


APO
-
BrdU


TUNEL Assay Kit (A
23210
)

Metabolic & nuclear changes: the end game

56

Vybrant
®

Apoptosis Assay Kit #
10
(V
35114
)

4 hours

camptothecin

Necrotic

(peroxide
-
treated)

APC annexin V

SYTOX
®

Green Dye

Resorufin fluorescence

C
12
-
resazurin

resorufin

Reductase


Allophycocyanin annexin V + C
12

resazurin + SYTOX
®

Green dye


Membrane asymmetry, metabolic activity and membrane integrity


488 & 633 nm excitation

A

B

C

D

Jurkat cells treated as labeled:
488
nm

&
633
nm

excitation

Live cells
,
annexin V
-
positive
,
SYTOX
®

Green
-
positive

Reductase activity / metabolic health

57

Chromatin Condensation / DNA Fragmentation

Vybrant
®

Apoptosis Assay Kit #
5
(V
13244
)

Hoechst
33342
/ propidium iodide

APO
-
BrdU
TM

TUNEL Assay Kit (A
23210
)

Alexa Fluor
®

488
anti
-
BrdU /

propidium iodide


Nuclear changes: the finale

58

59

www.invitrogen.com/iProtocol

60

www.invitrogen.com/iPath

61

www.invitrogen.com/Antibodies

62

63

64

Literature and brochures

Easy downloads from the web, or available on request.