Ask the “Experts” Tutorial

Apoptosis and Autophagy

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Jolene Bradford R&D Molecular Probes, Thermo Fisher Scientific

Karen Tamul EMD Millipore

Bill Telford National Cancer Institute National Institutes of Health

Apoptosis was first identified as a distinct morphological phenomenon in the 1960s (and probably earlier), and was well-accepted as an important regulatory process by the 1970s… apoptosis

cell shrinkage cytoskeletal collapse increased cell permability chromatin condensation DNA fragmentation transglutaminase crosslinking cell “blebbing”

necrosis

mitochondria still intact recognition and phagocytosis/ clearance

From Kerr, J.F.R., J Pathology 105, 13-20, 1971 (!)

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Signal transduction of apoptosis Signals

Extrinsic pathway

Intrinsic pathway

Death ligands (TNF, TRAIL, Fas ligand)

Stress, DNA damage Bax, Bam, Bid

Bid

TNFR1, Fas, DR4/5

Caspase activator Initiator caspase

FADD

Effector caspase

Morphology From Z. Darzynkiewicz

cytochrome C release

Smac/DIABLO

caspase 8, 10

Bcl-2, Bcl-xl

IAPs

Apaf-1 caspase 9

caspase 3, 6, 7 Autophagy Cytoskeletal breakdown Membrane asymmetry Chromatin condensation DNA fragmentation

Apoptosis

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Flow cytometry assays for apoptosis are now almost 25 years old… Cells were usually ethanol-treated (to permit loss of fragmented DNA) and labeled with cell cycle appropriate concentrations of a DNA binding dye (usually propidium iodide)

changes in light scatter

the “sub-G0/G1” peak

Apoptosis measurement in individual cells (not lysates)

Analysis on an Ortho Cytofluorograph 50-H (!)

From Telford et al., Applied Fluorescence Technology 4, 12-17 (1992)

Types of assays… unfixed “viable” cells “early” apoptotic events

“late” apoptotic events

fixed permeablized cells

Cell volume fluctuations Changes in cell membrane potential Mitochondrial potential changes Signaling events (bcl-2, Bax, etc.) Initiator (proximal) caspase activation (1,9,10,8)

Cytochrome C release

Effector (distal) caspase activation (3,6,7)

Effector (distal) caspase activation

Organelle changes PS membrane “flipping” Transglutaminase crosslinking Membrane “blebbing” Loss of membrane permeability

Initator (proximal) caspase activation

Changes in chromatin organization (histones) Early DNA strand breaks Global chromatin damage

We won’t directly cover the more common apoptosis assays today (DNA dye permeability, annexin V, TUNEL), but will concentrate on earlier signaling/ effector assays such as caspase activation.

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Take-home lessons… Apoptosis is a highly variable process. There is a lot of variation in the apoptotic process and phenotype between cell types, and even the same cell type at different levels of activation or differentiation. You therefore need to find the best method for measuring apoptosis for your particular cell system. Don’t just choose a method at random! Never use only one assay for apoptosis. And combine multiple assays wherever possible! Always measure cell death using several different methods, preferably in the same sample. Multiparametric flow cytometry is ideal for this. Let your assay not only measure cell death, but characterize it as well. You can learn interesting things about your cells and your system. Take pictures! Visualizing the cells is important and very educational! Many new options in image cytometry make this possible. PreCongress Tutorial

Using DNA dyes and viability probes as a starting point for apoptosis assay DNA dyes used as viability probes (like PI, 7-AAD and DAPI), and covalent viability probes like Live/Dead (Life Tech) and the Zombie dyes (BioLegend) are great starting points for building an apoptosis assay. Alone, they are not enough to “prove” apoptosis, but they make essential counter-labels for assays like annexin V and caspase substrates.

EL4 cells cycloheximide

Live/Dead Long Red (EM ~ 670 nm)

EL4 cells cycloheximide

7-aminoactinomycin D

propidium iodide

DNA dyes are NOT fixable – covalent viability probes ARE. L1210 cells anti-Fas Ab

DC1107

forward scatter

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Using DNA dyes and viability probes as a starting point for apoptosis assay The Live/Dead dyes (Life Tech) come in a variety of colors. Pick one with minimal Spectral overlap into your apoptosis. Near IR (red laser excited) and Violet, Aqua and Yellow (violet laser excited) are good choices. The BioLegend Zombie dyes work by the same principle, and are also available in Long Red, Aqua and Yellow versions. L1210 cells anti-Fas Ab

Zombie Aqua

Zombie Yellow

EL4 cells camptothecin

forward scatter

forward scatter

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Combining multiple apoptosis assays Annexin V and a DNA binding dye is an excellent example of combining two assays into a multiparametric method. EL4 cells actinomycin D 4 hours propidium iodide

PS “flipping” occurs prior to 7-AAD permeability

16%

annexin V+ PI+ (late apoptotic/ necrotic) 75.1%

annexin V+ PI“early apoptotic” 7%

“viable” cells

At least two stages of apoptotic death are being measured here.

FITC annexin V PreCongress Tutorial

Interpreting forward and side scatter Many cell types show a distinctive loss of forward scatter and increase in side scatter during apoptosis. However, this is not always the case. Fixation can also blur the distinction between live and dead cells. Gate carefully! We recommend that you gate on both “viable” and “non-viable” cells, but look at the apoptotic phenotype in the scatter”viable” cells only as well. You will be able to see the earliest stages of apoptosis.

side scatter

EL4 cells camptothecin paraformaldehyde fixed

L1210 cells Anti-Fas Ab unfixed

EL4 cells camptothecin unfixed all cells scatter “viable”

forward scatter

DC1110

Combining multiple apoptosis assays With scatter, four parameters are being measured here. Combining assays allows the progression of apoptosis to be studied, and provides a much richer picture than any one assay can give.

7-amnoactinomycin D

side scatter

mouse L929 cells TNFa + cycloheximide 4h

scatter “viable” cells

all cells

mouse L929 cells no treatment

63.9%

13.6%

20.2%

53.8%

8.9%

24.3%

FITC annexin V

Annexin V binding precedes both scatter changes and 7-AAD incorporation. forward scatter

We are looking at a relatively early apoptotic event compared to other assays.

Caspase substrates

Caspase substrate peptides coupled to a fluorochrome, and in some cases a reactive group. Three major types… FLICA

Fluorochrome-labeled inhibitors of caspases

PhiPhiLux

Exciton-based fluorogenic caspase substrates

CellEvent Green NucView 488

Substrate-immobilized DNA binding dye

All can be used to analyze endogenous caspase activity in unfixed cells. All have advantages and limitations.

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FLICA substrates Available from a number of commercial sources Immunochemistry Technologies FLICA Life Tech Invitrogen Vybrant FAM and Image-iT

fluorescein or sulforhodamine

Consists of a caspase consensus substrate peptide flanked by a fluorochrome molecule and a fluoromethylketone (FMK) reactive group.

substrate peptide

Complex is always fluorescent. Unfixed cells are incubated with the substrate, which interacts with active caspase binding domains. The FMK regions then crosslinks to amine residues at the caspase activation site. FLICA reagents are inhibitors, since they crosslink and inactivate caspases.

fluoromethylketone (FMK) reactive group

The unreacted substrate is then washed out. Cells can be analyzed as is, or fixed for later analysis. Available in fluorescein, sulforhodamine 101 and Cy5 (FLICA 660) conjugates. In vivo form available (FLIVA). PreCongress Tutorial

FLICA detection of apoptosis-associated caspase activation The FLICA reagents can (and should) be combined with a DNA permeability dye, like propidium iodide or 7-AAD.

3.01%

cycloheximide

Hoechst 33258

cycloheximide

no treatment

no treatment

48.4%

forward scatter

FLICA FL caspase 3/7

FLICA detection of apoptosis-associated caspase activation The FLICA reagents can be combined with annexin V and a DNA dye like 7-AAD for a powerful multiparametric apoptosis assay. Cells should not be fixed. EL4 cells no treatment

FLICA

early apoptotic

“viable”

late apoptotic “viable”

late apoptotic

side scatter

topotecan 2 μM 16 h

early apoptotic

DC1125

forward scatter

7-AAD (SYTOX AADvanced)

Pacific Blue annexin V

FLICA detection of apoptosis-associated caspase activation The FLICA reagents can also be combined with covalent viability dyes like Live/Dead. Cells can be fixed under these circumstances. no treatment

topotecan 2 μM 16 h

side scatter

EL4 cells

Live/Dead Near IR

forward scatter

DC1125

FLICA

FLICA 660 A red-excited, red-emitting FLICA reagent (FLICA 660) is available from Immunochemistry Technologies, Inc. EL4 cells

no treatment

topotecan 2 μM 16 h

cell number

Zombie Yellow

Gated on all cells

DC1119

FLICA 660 caspase 1

FLICA 660 caspase 1

FLICA 660 poly caspase

FLICA substrates Advantages FLICA substrates covalently crosslink to the active site, so location of caspase activity is retained. Fixed and permeablized cells can be analyzed at a later time.

Issues FLICA substrates have been found to bind non-specifically to intracellular sites with no caspase activity (FMK problems).

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PhiPhiLuxTM caspase substrates Oncoimmunin, Inc. • peptide backbone containing enzyme consensus cleavage site (DEVD for caspase 3/7) • fluorochrome molecules attached to terminal ends of the peptide • peptide backbone modified to bring fluorochrome molecules into close steric proximity, resulting in fluorescent quenching • complex is relatively non-fluorescent when uncleaved • cleavage of the consensus site “frees” the fluorochromes, which then fluoresce • cells are not permeablized or fixed following substrate incubation, but are analyzed immediately – PhiPhiLux does not inactivate caspase, and is not an inhibitor • conjugated with fluorescein-, rhodamine and Cy5-like fluorochromes

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PhiPhiLux caspase substrates Argon-ion 488 nm

Like annexin V, PhiPhiLux can (and should ) be combined with a DNA binding dye (PI or 7-AAD are fine). late apoptotic/ necrotic

caspase 3

7-AAD

all cells

7-AAD

intermediate caspasepositive apoptotic

early caspasepositive apoptotic

“viable” cells

7-AAD negative

fluorescein PPL caspase 3 fluorescein PPL caspase 3 EL4 cells cycloheximide 50 µg/ml 4 hr

PhiPhiLux caspase substrates DPSS 488 nm

EL4 cells labeled with fluorescein PhiPhiLux, APC-conjugated annexin V and 7-AAD

caspase 3 7-AAD

HeNe red or red diode 633 nm

APC-annexin V

Caspase activation is early (before annexin V binding and 7-AAD permeability ungated

actinomycin D 8h

forward scatter

7.4%

APC-annexin V

side scatter

no treatment

7-AAD negative annexin V negative gated

7.1%

2.8%

52.5%

18.4%

47.9%

7-AAD

fluorescein PPL caspase 3

Detection of two distinct caspase activities by flow cytometry

caspase 8

Argon-ion 488 nm

PE-annexin V 7-AAD

Simultaneous labeling of apoptotic EL4 cells with fluorescein and Cy5 caspase 3 and 8 substrates

Red diode 635 nm

caspase 3

caspase 8 (pro-caspase 3)

HeNe red 633 nm

Ar-ion 488 nm

caspase 3

350

400

450

500

550

600

Cy5 PPL caspase 3

4.1%

650

Cy5

68.0% 22.8%

R2D2 only

L1D2 only

Relative fluorescence intensity

fluorescein

AnnV-neg 7-AAD-neg fluorescein PPL caspase 8 350

400

450

500 550

600 650

Wavelength

700 From Telford, W.G., Komoriya, A. and Packard, B.Z. (2004) Methods in Molecular Biology Volume 263, Flow Cytometry Protocols, pp. 141-159.

GemstoneTM probability state modeling of caspase activation Simultaneous labeling of apoptotic EL4 cells with fluorescein and Cy5 caspase 3 and 8 substrates Our “knowns” are forward and side scatter, propidium iodide membrane permeability and caspase 3. Layering in caspase 8, we can see that it comes up well before caspase 3. Gemstone analysis by C. Bruce Bagwell, Verity Software House

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Detection of three distinct caspase activities by flow cytometry DPSS greenyellow 561 nm

Ar-ion 488 nm

DPSS 488 nm

fluorescein caspase 8

fluorescein caspase 8

Near-UV diode 375 nm

Hoechst 33258

no treatment 350

400

450

500

550

450

500 550

600

650

700

Cy5 caspase 3

350

400

rhodamine caspase 1 Cy5 caspase 3

TAMRA caspase 1

600 650

700

Wavelength DPSS 561 nm excitation should permit simultaneous excitation of rhodamine and Cy5 labeled substrates.

Hoechst 33258 exclusion

Relative fluorescence intensity

DPSS green-yellow 561 nm

cycloheximide 4h

0.8%

rhodamine caspase 1

22.8%

forward scatter

caspase 1 caspase 8 (pro-caspase 3)

caspase 3

Cy5 caspase 3

Analyzing multiple substrates by flow cytometry allows us to temporally map caspase activation in intact cells. This is no longer just an apoptosis assay, but is defining how apoptosis occurs.

E2D2 caspase 1 only

8,1 -pos 3 -pos

3-R2D2 caspase 3 only

rhodamine caspase 1

Hoechst 33258

rhodamine caspase 1

Detection of three distinct caspase activities by flow cytometry

FL caspase 8

8,1 -pos 3 -pos

8,1 -pos 3 -pos 8,1 -pos 3 -neg

8, 1 -pos 3 -neg

L1D2 caspase 8 only

FL caspase 8

E2D2 caspase 1 only

rhodamine caspase 1

GemstoneTM probability state modeling of caspase activation We then add in caspase 1, and show that is comes up early, about the same time as caspase 8. Backgating to scatter shows some interesting scatter transitions early in the apoptotic process.

Gemstone analysis by C. Bruce Bagwell, Verity Software House

Detection of four distinct caspase activities by flow cytometry

Solid state 488 nm

FITC caspase 1 Red diode 635 nm

Cy5 caspase 3

E1D2 caspase 1

forward scatter

With a multi-laser flow cytometer, we can distinguish up to four distinct caspase activities simultaneously, and determine their activation order. 1

Green DPSS 532 nm

Hoechst 33258

8

1 3- 1+ 8+

3- 1+ 8-

caspase 3 negative Hoechst 33258

6 3- 1+ 6-

3- 1+ 6+

J6D2 caspase 6 J6D2 caspase 6

R2D2 caspase 3

3

TAMRA caspase 6

UV 355 nm

forward scatter

1,6,8

SR101 caspase 8

8

6

3- 6+ 8+

3- 6- 8+

caspase 1 caspase 8 (pro-caspase 3) caspase 6 caspase 3

L2D2 caspase 8

This is no longer just an apoptosis assay. We are observing the signal transduction of apoptosis.

Detection of four distinct caspase activities by flow cytometry

GemstoneTM analysis

caspase 1 caspase 8 (pro-caspase 3) caspase 6 caspase 3

Again, this is no longer just an apoptosis assay. We are observing the signal transduction of the apoptotic process, and doing it in a relational and multiparametric way (as only flow cytometry can!). Gemstone analysis by Beth Hill, Verity Software House

PhiPhiLux caspase substrates Advantages They are relatively non-fluorescent prior to cleavage, making for lower backgrounds. Caspases remain active, since the substrate does not crosslink and inactivate the enzyme. Specificity studies demonstrate relatively good specificity for target caspases.

Issues They do not covalently bind to the site of activity – analysis and localization studies must be done quickly. Not fixable. The cleaved form will diffuse out of the cell over time.

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NucView 488TM (Biotium) CellEvent GreenTM (Life Technologies) A substrate complex that binds to DNA upon enzyme cleavage Loaded into viable cells – in the presence of caspase, the complex is cleaved and the dye can bind to nuclear DNA. Not fixable. Gated on scatter-viable cells

no treatment

Seems to be very rapid for loading (15-30 minutes incubation time)

7-AAD

Should not be fixed.

cycloheximide

Nuc 488 (fluorescein)

No site specificity – probably better for rapid screening

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CellEvent GreenTM (Life Technologies) Like FLICA and PhiPhiLux, CellEvent Green can be combined with annexin V and a DNA binding dye. EL4 cells

side scatter

topotecan 2 μM 16 h

Pacific Blue annexin V

7-AAD

no treatment

forward scatter

CellEvent Green

CellEvent GreenTM (Life Technologies) CellEvent Green can also be combined with covalent viability dyes like Live/Dead. Cells should NOT fixed under these circumstances. no treatment

topotecan 2 μM 16 h

side scatter

EL4 cells

Live/Dead Near IR

forward scatter

CellEvent Green

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NucView 488TM (Biotium) CellEvent GreenTM (Life Technologies) Advantages Rapid incorporation and labeling, and washing not required.

Issues They do not covalently bind to the site of activity – analysis and localization studies must be done quickly. Not fixable. Not site-specific.

About all caspase substrates… No synthetic substrate is completely specific for its target enzyme. Cell permeability is never total. Can caspases be activated in circumstances other than apoptosis?

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Do PhiPhiLux and FLICA agree with one another? Co-labeling with PhiPhiLux and FLICA directed against the same caspase

DPSS 488 nm

PPL or FLICA DPSS green-yellow 561 nm

PhiPhiLux (non-inhibitory)

PPL rhodamine or FLICA SR101

FLICA FL caspase 3

G1D2 (FL) caspase 3

Hoechst 33258

PPL rhodamine caspase 8

forward scatter

FLICA SR101 caspase 3

side scatter

PPL rhodamine caspase 3

FLICA (inhibitory)

forward scatter

FLICA FL caspase 8

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Caspase substrate specificity

PhiPhiLux

FLICA

Jurkat T cells

control

staurosporin

Caspase substrate specificity

PhiPhiLux

FLICA

Jurkats

control

staurosporin

Caspase substrate specificity

PhiPhiLux

FLICA

Jurkat T cells staurosporin 1 µM 4 h

time of substrate incubation

10 min

20 min

FLICA binds to apoptotic cells almost immediately following addition, suggesting that caspase-dependent interactions may not be the primary mechanism of FLICA labeling of apoptotic cells

40 min

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Immunodetection of cleaved caspase 3 by flow cytometry Several monoclonal antibodies against the cleaved active form of caspase 3 are available (BD Biosciences rabbit monoclonal and Cell Signaling Technologies polyclonal rabbit) conjugated to several fluorochromes and biotin.

camptothecin

side scatter

control

forward scatter

These antibodies can label apoptotic cells in fixed cell preparations. Requires paraformaldehyde fixation and detergent treatment.

PE anti-cleaved caspase 3 (BD)

Saponin-based methods of the type used for intracellular cytokine analysis work well too. FITC anti-cleaved caspase 3 (BD)

Immunodetection of cleaved caspase 3 and Live/Dead labeling

Near IR Live/Dead viability

Since caspase 3 immunolabeling requires fixation permeablization, you can combine it with a fixable Live/Dead assay in place of a DNA binding dye. Cells should be labeled with the Live/Dead reagent prior to fixation.

untreated

camptothecin 5 μM 16 h “late” apoptotic

“early” apoptotic “viable” cells

PE anti-cleaved caspase 3

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Immunodetection of cleaved caspase 3 and TUNEL Since caspase 3 immunolabeling requires permeablization, you can combine it with a TUNEL assay for an even better multidimensional picture of apoptosis in fixed cells. Cells were TUNEL labeled, then caspase labeled.

caspase 3 only

PE anti-cleaved caspase 3

4.8%

As expected, caspase 3 comes up first, but only slightly precedes DNA fragmentation.

63.4%

“late” caspase 3 -positive DNA fragmentation -positive 31.2%

“early” caspase 3 -positive DNA fragmentation -negative TUNEL only

DC324

Fluorescein dUTP TUNEL

“viable” cells

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Do substrate and immunolabeling agree with one another? PhiPhiLux and caspase 3 immunolabeling? Apoptotic EL4 cells were labeled with PPL and sorted into caspase-negative and caspase-positive fractions. sorted cells

unsorted cells

PPLPPL negative negative sorted cellscells sorted

side scatter

unsorted apoptotic cells

forward scatter

25.2%

immunolabel for cleaved caspase 3

1.3% 2.2%

unsorted cells

PPL caspase negative sorted cells

sort PPL positive sorted cells

fluorescein PPL caspase 3

97.9%

PPL caspase positive sorted cells

PE-anti-cleaved caspase 3

Caspase activity measured by PhiPhiLux substrate cleavage and by immunolabeling correspond very precisely.

Do substrate and immunolabeling agree with one another? Since FLICA is fixable, it can also be combined with anti-active caspase 3 antibody, both as a more robust assay and to see if the two caspase assays “agree” with each other.

PE anti-active caspase 3

Cells were labeled with FLICA, fixed and labeled with anti-active caspase 3. L1210 cells anti-Fas Ab

EL4 cells camptothecin

no label FLICA only anti-active caspase 3 only

FLICA

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Do substrate and immunolabeling agree with one another? Since FLICA is fixable, it can also be combined with anti-active caspase 3 antibody, both as a more robust assay and to see if the two caspase assays “agree” with each other.

PE anti-active caspase 3

Cells were labeled with FLICA, fixed and labeled with anti-active caspase 3. L1210 cells anti-Fas Ab

EL4 cells camptothecin

no label FLICA only anti-active caspase 3 only both

FLICA Very slight disagreement! It might be caspase 7 (which the antibody does not see), or cells that have undergone apoptosis between FLICA and antibody labeling.

Apoptosis and image cytometry Visualizing apoptotic cells is an excellent idea. Why? Apoptosis is highly variable and pleiotropic. Imaging can give verification that apoptosis is occurring, and characterize it. Imaging gives additional analysis options (like pixel-by-pixel analysis) that are useful for apoptotic analysis. Imaging allows analysis of adherent cells without removal of the cells from their substrate. Many options now exist for performing image cytometry, where cytometric data and correlated cell images can be collected simultaneously. Many laboratories prefer this technology for the analysis of apoptotic cells (particularly for adherent cells).

viable cell

apoptotic cell

Compucyte iCys

Compucyte iCys analysis of apoptotic EL4 cells no treatment

no treatment Alexa Fluor 647 annexin V

Apoptosis and image cytometry

7-AAD

7-AAD viable early apoptotic advanced apoptotic

fluorescein PPL caspase 3

camptothecin 6 h

Alexa Fluor 647 annexin V

camptothecin 6 h

7-AAD

7-AAD

Compucyte iCys field scans

viable early apoptotic advanced apoptotic

fluorescein PPL caspase 3

Apoptotic cell analysis with laser scanning cytometry Alexa Fluor 647 annexin V

Direct correlation between the cytometric data and the imagery (relocation analysis). Morphological analysis using light scatter or absorption.

fluorescein PPL caspase 3

Discrimination of adherent apoptotic cells by image cytometry AF647 AnnV + caspase 3

caspase 3

Hoechst 33258 PhiPhiLux caspase 3 Alexa Fluor 647 annexin V

UMR-106 cells Compucyte iCys field scan

Image cytometry can analyze site-specific fluorescence from images, improving sensitivity. Trypsin or accutase detachment, which can “muddy” apoptotic labels, is not necessary. Apoptotic cells “round up” and can be lost from the growth substrate.

AF647 annexin V

Max Pixel versus total Integral

PPL caspase 3 Max Pixel

Max Pixel

Integral PPL caspase 3

Analyzing apoptosis on the Amnis ImageStream A stream-based scanning cytometry system like the Amnis ImageStream or FlowSight similarly is an another excellent way to combine cytometric and morphological analysis.

Brightfield

Composite

Again, direct correlation between cytometry and imagery. Daudi cells induced with camptothecin Brightfield

Phi Phi Lux

Annexin V PB

Draq5

Composite

Data from Brian Hall and Tad George, Amnis

Analyzing apoptosis on the Amnis ImageStream camptothecin 6 h

3%

fluorescein PPL caspase 3

fluorescein PPL caspase 3

untreated

70%

Brightfield Phi Phi Lux Annexin V Draq5 Composite

Brightfield Phi Phi Lux Annexin V Draq5 Composite

Images of live cells

Images of PhiPhiLux positives Data from Brian Hall and Tad George, Amnis

Analyzing apoptosis on the Amnis ImageStream Phi Phi Lux

Annexin V

Draq5

Composite

Intermediate apoptotic

Late apoptotic

10%

Brightfield

Phi Phi Lux

Annexin V

Draq5

Composite

Pacific Blue annexin V

camptothecin 6 h

Annexin V Intensity

Brightfield

Brightfield

Phi Phi Lux

Annexin V

Draq5

Composite

Brightfield

Phi Phi Lux

Annexin V

Draq5

Composite

77%

PPL caspase 3

Viable and very early apoptotic

Early apoptotic

Data from Brian Hall and Tad George, Amnis

Analyzing apoptosis on the Amnis FlowSight in-focus events PacificV_Pac BlueBlue annexin Annexin Intensity V

1e6

A lower cost platform for simultaneous correlated cytometric and image analysis.

EL4 cells CPT

1e5

1e4

late apoptotic 1e3

early apoptotic 0

1e3

1e4

7AAD Intensity

7-AAD

1e5

viable

viable

early apoptotic

late apoptotic Data from Darin Fogg, Amnis

Types of assays… unfixed “viable” cells “early” apoptotic events

“late” apoptotic events

fixed permeablized cells

Cell volume fluctuations Changes in cell membrane potential Mitochondrial potential changes Signaling events (bcl-2, Bax, etc.) Initiator (proximal) caspase activation (1,9,10,8)

Cytochrome C release

Effector (distal) caspase activation (3,6,7)

Effector (distal) caspase activation

Organelle changes PS membrane “flipping” Transglutaminase crosslinking Membrane “blebbing” Loss of membrane permeability

Initator (proximal) caspase activation

Changes in chromatin organization (histones) Early DNA strand breaks Global chromatin damage

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Simultaneous immunophenotyping and apoptosis assays Why we all used flow cytometry for apoptosis detection in the first place! But, is it feasible? Remember, apoptotic cells are killing themselves. And as most cytometrists know from painful experience, dead cells bind high levels of antibodies nonspecifically (that’s why we use PI). Therefore, it is risky. But if you are determined to do it… Don’t try it with assays detecting late manifestations of apoptosis (like sub-G0/G1 or loss of membrane permeability). Much too late. Even annexin V is pushing it. Use early assays, like proximal caspase activation. They combine readily with fluorescent immunophenotyping.

Interpret data with extreme caution! PreCongress Tutorial

So what assay is best for my application? That depends on your application. What question are you asking? Example: Yes-no viability screening of a new drug. Many samples, high throughput. Fixed assays (“sub-G0/G1”, TUNEL, FLICA, caspase immunolabeling) will be the most practical for large numbers of samples where they will have to sit for a while prior to analysis. Simple is probably better, although a two-parameter assay is best to exclude necrotic cells.

Example: Analyzing ability of lymphocytes to undergo apoptosis in a signal transduction knockout mouse model. Small numbers of samples. A different question. You don’t just want to quantify apoptosis, you want to characterize it, especially at the signaling level. A combination of “viable” and fixed cell assays to analyze caspases, caspase targets and other apoptotic signaling molecules. Multiple assays within a single sample will give the most information.

Don’t limit yourself to one method! Combine whenever possible.

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So what assay is best for my cells? Apoptosis is a highly variable process. Determine what assays work best for your cell system. Understand how your cells undergo apoptosis and design your detection method accordingly. Example: EL4 cells treated with cycloheximide (transcriptional inhibitor) Strong caspase 3 expression High levels of DNA strand breaks No blebbing (annexin V binding strong)

Example: MCF-7 cells with ellipticine (topoisomerase II inhibitor) Undetectable levels of caspase 3 expression Activation of caspase 8 and 9 Blebbing (“sub-G0/G1” peak detection and annexin V detection problematic) High levels of DNA strand breaks

Don’t limit yourself to one method! Combine whenever possible.

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