measuring apoptosis in real time with a new luminescent method

Measuring Apoptosis in Real Time by Linking

Luciferase Fragments to Annexin V

Mourad FERHAT, Ph.DProduct Manager Cellular Analysis and Proteomics

FDSS Users Meeting – June 8th, 201713th European Functional Drug Screening Symposium

2Promega CorporationPromega Corporation©2015 Promega Corporation.

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Who we are

Manufacturer of reagents, kits and integrated systems for life science market

Promega Headquarters Madison, WI

1,300 employees in 15 countries

Over 3,500 products ~ 750 patents

Key technologies :

Cellular Analysis

Genomics

Integrated Solutions

Custom Assay Service :

Cell engineering

Assay developmentThe Feynman center cGMP facility (260,000 square feet, Madison for IVD manufacturing


Promega in cellular analysis

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Cell Health

Cell Signaling

Metabolism

Protein Function

• Cell Viability

• Cytotoxicity

• Apoptosis

• Oxidative Stress

• Pathway Analysis

• GPCR

• Nuclear Receptor

• Kinases assays

• Glucose Uptake

• Lactate

• Glutamine/Glutamate

• Glycolysis

• Protein Localization and trafficking

• Protein:Protein Interactions

• Biochemical Assays

Drug Discovery• Potency assays (ADCC, ADCP)

• Immune checkpoints

• Target Engagement

Imaging • Live-cell imaging

• In vivo Imaging

Real-Time Apoptosis

AutophagyGasAC

cAMP

PK

A

ATP

ATPATPATP

LIVE CELL

PROTEASE

DEAD CELL

PROTEASE

ProCaspase-3Caspase-3

GSHGSSG

P450

P450

N

UGT


Promega in Drug Discovery

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Bioassays

Cell Health In vivo imaging

Cell signaling

Ab characterization

Ab purification

ADMECell metabolism

Target engagement

Bioassays :

ADCC, ADCP

Immunecheckpoints

T-cell Activation Bioassays

Cytokines reporter cell lines

Outline

Promega Corporation 5

• Apoptosis in Health and disease

• Cell membrane changes during apoptosis

• Annexin V

• Fluorescent Annexin V binding assay

• How luminescent Annexin v binding assays work

• Example data

• Summary


Apoptosis in Health and disease

• 50-70 million cells die by apoptosis

everyday in the average human adult

(~0.5%).

• Apoptosis is an important homeostatic

mechanism to eliminate cells that have

irreparable DNA damage, are virally

infected or are actively targeted by the

immune system.

• Dysregulation of the apoptotic process

can lead to cancer (insufficient) or

degenerative diseases such as (AIDS or

Parkinson’s (excessive).


Studying Apoptosis In Vitro

Viable/Proliferating Morphological Changes “Blebbing” and 2°Necrosis

Apoptotic ProgressionUntreated

Stimulus

30 minutes to 48hrs• Dependent upon strength of stimulus• Dependent upon mode of action

The caspase enzyme cascade is an active participantExposure and Dose!


Phosphatidyl serine exposure during Apoptosis

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Normal Cell - Viable Apoptotic Cell - Viable 2° Necrosis Non-Viable

Phosphatidyl Serine

Time following exposure to apoptosis inducer

PS confined to inner leaflet

Cell membrane intact

PS flipping to outer leaflet


PS on outer leaflet and inside cell

Cell membrane compromised

Promega Corporation©2016 Promega Corporation.

- Reutelingsperger et. al. demonstrated that cells undergoing apoptosis actively translocate their PS from inner leaflet to the outer membrane prior to losing membrane integrity


Annexin V as a marker of Apoptosis

Phosphatidylserine (PS) head groups are normally oriented

toward the cytoplasmic side of membrane of viable cells

• During apoptosis PS headgroups“flip” to outer leaflet

• Annexin V is a Ca2+- dependent PS binding protein

• Annexin V binding to intact cells indicates apoptosis


Progression from normal to apoptosis to secondary necrosis

Annexin V binds to PS on the outside and inside of necrotic cells (right). DNA staining also

occurs in necrotic cells (right).

Fluorescent Annexin V-FITC Binding Assay

• Annexin V is chemically conjugated to a fluorophore for detection of apoptosis

• Must wash cells in Ca++ buffer (can lose apoptotic ones!) then apply AnV-FITC


Annexin V binds to surface exposed PS on apoptotic cells (center)

and PS in cytoplasm in necrotic cells. Dual staining with annexin and

DNA dye indicates necrosis (right).

… After Wash Step, Analyze by Flow Cytometry

Annexin (-), PI (-) Annexin +, PI (-) Annexin +, PI +

Washes = extra time and effort!

Washes can be detrimental to cell health when that’s what you’re measuring!

Flow cytometry:

1. Requires trypsinizationof attached cells for analysis. Trypsin can be harsh on cell health.

2. Requires special instrument and userexpertise.

3. Low throughput!


Configuring NanoLuc into a binary reporter

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156 aa

11aa

NanoLuc® Binary Technology(NanoBiT®)

+

LgBiT17.6 kDa

SmBiT11 aa

Protein A Protein BA:B

interaction

Structural complementation

gives a bright, luminescent enzyme


How the homogeneous Luminescent Annexin V

Binding Assay works ?

• Annexin V-luciferase fragment

fusion proteinsAnnexin V - Small fragment

Annexin V - Large fragment

• Fusion proteins (by themselves

or when combined) with viable

cells do not produce light

• When annexin-fusion proteins

bind to PS in close proximity on

the surface of apoptotic cells, the

SmBiT and LgBiT fragments

reconstitute to form an active

luciferase enzyme

• CellTox Green DNA binding dye

(Necrosis Detection Reagent)

detects 2° necrosis

+

LgBiT17.6 kDa

SmBiT11 aa

Annexin V Annexin VAnxV:AnxVinteraction

Structural complementation

gives a bright, luminescent enzyme


NanoLuc

Homogeneous Real Time Apoptosis Assay

Using Annexin-NanoBiT Binding to PS

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Normal Cell - Viable Apoptotic Cell - Viable 2° Necrosis Non-Viable

Annexin V Annexin V

LgBiTSmBiT

Phosphatidyl Serine

Necrosis

Detection

Reagent

Time following exposure to apoptosis inducer

PS confined to inner leaflet


Low luminescence

Low fluorescence

PS flipping to outer leaflet


High luminescence

Low fluorescence

PS on outer leaflet and inside cell

Cell membrane compromised

High luminescence

High fluorescence

Promega Corporation©2016 Promega Corporation.


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Real Time Apoptosis and Secondary Necrosis

Detection

The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay reagent was added once at time zero

to DLD-1 cells treated with rhTRAIL. Luminescence (RLU) and fluorescence (RFU) were recorded repeatedly from the same samples.

Multiplexing real-time apoptosis and secondary necrosis detection of DLD1 cells treated with rhTRAIL clearly indicates the timing of induction of apoptosis.


The Homogenous RealTime-Glo™ Annexin V

Assay Reagent

Annexin V Large BiT (1000x)Annexin V Small BiT (1000x)RT-Glo Annexin Substrate (1000x)Necrosis Detection Reagent (1000x)CaCl2 (1000x)

Create 2x Reagent by dilutionin their cell culture medium

What we provide

Seed Cells Dose cells Add Reagent Incubate for exposureMeasure RFU and RLU (repeat)

No washes, simple measures!


Comparison of Annexin Protocols

Fluorescent

Annexin V-FITC

+ PI

Incubate Wash

away unbound

fluorescence

Flow cytometry

Luminescent

Annexin V-NanoBiT fusions

+ Necrosis Detection Reagent

Incubate

Plate reader

• No wash step

• Can read the same samples

multiple times


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U2OS untreated / TRAIL/ Bortezomib

control20x/0.7

TRAIL 200 ng/ml, 8h

150x

Bortezomib, 1 M, 24h


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Plate-Based Natural Killer (NK) Activity Assay

Sensitivity (2hr) Kinetic Mode

NK

Target (K562)


Time : Minute 1-240Killer : Target Ratio 2:1

Bioluminescent imaging of natural killer cell

function : NK-92mi Induced Cell death of K562 cells

Bright Field

Bioluminescence

90minTIME: 5min 180min

Initial Engagement Granzyme Delivery Target Cell Apoptosis

NK

Target

19Promega Corporation

Summary of Annexin-NanoBiT Assay Approach

• Homogeneous single reagent assay with no wash steps

• Data recorded with standard multimode plate reader

• Enables “real time” data recording to monitor kinetics of

apoptosis and secondary necrosis in the same sample

• Samples can be used for additional downstream

applications (e.g. multiplexing with other assays)

measuring apoptosis in real time with a new luminescent method

Science