We believe this site might serve you best:

United States

United States

Language: English

Promega's Cookie Policy

Our website uses functional cookies that do not collect any personal information or track your browsing activity. When you select your country, you agree that we can place these functional cookies on your device.

RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay

JA1011

Simple, Plate-Based Assay for Real-Time Apoptosis Detection

  • No-wash, one-step Annexin V binding assay
  • Non-lytic. Allows continual monitoring of cell state to accurately determine apoptotic onset
  • Scalable for high-throughput screening applications

Choose an assay

Size

Catalog number selected: JA1011

$ 495.00 Your price: Log in

Request a sample Add to Helix Add to Helix Add to Helix Add to Helix
RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay
Apoptosis and Necrosis Assay/100 assays
$ 495.00
Your price: Log in
Change Configuration
36524203-annexinv-overviewV2

Real-Time Monitoring of Apoptosis Progression

The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay measures the real-time exposure of phosphatidylserine (PS) on the outer leaflet of cell membranes during the apoptotic process. Annexin V luciferase fusion proteins supplied in the assay reagent bind to PS during early apoptosis and are detected with a simple luminescence signal. The reagent also includes a DNA-binding dye, which enters the cell and generates a fluorescent signal upon loss of membrane integrity.

The combination and timing of luminescent and fluorescent signals is used to differentiate secondary necrosis occurring during late apoptosis from necrosis caused by other cytotoxic events.

The assay is non-lytic and the simple “add-and-read” method allows multiple readings from a single assay well. Apoptosis can be monitored in real time, without the need for multiple plates, complicated processing, or specialized detection equipment. A multimode reader capable of detecting luminescence and fluorescence is the only instrument required.

 

How the Assay Works

Based on NanoLuc® Binary Technology (NanoBiT), the Realtime-Glo™ Annexin V Apoptosis Assay uses Annexin V fusion proteins containing complementary subunits of NanoBiT® luciferase (Annexin V-LgBiT and Annexin V-SmBiT). When in close proximity, the LgBiT and SmBiT subunits come together to form a functional luciferase.

Healthy Cell

The Detection Reagent contains  Annexin V-LgBiT and Annexin V-SmBiT (NanoBiT) fusion proteins and a profluorescent DNA dye.

14332MA-W-a Annexin V protein NanoBit fusions are bound to phosphatidylserine

Early Apoptosis

Luminescence remains low until PS exposure brings the annexin fusion proteins close together, forming a functional luciferase.

14332MA-W-b Phosphatidylserine rafts on the apoptotic cell surface bind Annexin V

Secondary Necrosis

A fluorescent signal is generated upon loss of membrane integrity during late stage apoptosis, when the DNA dye can enter the cell.

14332MA-W-c Membrane rupture in secondary necrosis is indicated by fluorescence

What the Readout Looks Like

Measurement of luminescence and fluorescence repeatedly from the same sample well after exposure to a test compound allows resolution of the effects of cytotoxic stimuli and determination of mechanism of action. Here, the time delay between the emergence of PS:Annexin V binding and the loss of membrane integrity indicates an apoptotic phenotype that leads to secondary necrosis. 

14275MA-W-a Annexin V Apoptosis and Necrosis Assay readout

Add One Reagent, then Read Repeatedly

Different Doses, Multiple Time Points, One Assay Plate

With this real-time assay, repeated measures of luminescence and fluorescence from the same well during compound exposure allow determination of compound potency using far fewer plates and less reagents than endpoint assays. 

The reagent is well tolerated by a variety of cultured cell types and can be applied prior to treatment or at dosing for real-time measurement of the dose and time-dependent magnitude of apoptotic progression. 

RealTime-Glo™ Apoptosis Assay

Multiple data points. One reagent addition. One assay plate.

14344MA-W-aV2

Data for each dose was obtained at multiple timepoints using a single assay plate with one reagent addition.

Endpoint Assay

Multiple date points. Multiple assay plates.

14344MA-W-bV2

Here, 7 plates were needed to obtain the data that could be achieved using a single plate with the real-time assay.


See More Data: Learn more about this data and see how the RealTime Apoptosis Assay can be used with various cell models exploring safety and exposure in the poster  A Real-Time, Bioluminescent Annexin V Assay for the Assessment of Apoptosis, presented at SOT 2017. 

Results are consistent with those from fluorescent annexin binding methods detected by flow cytometry.

36565334-RealTme-Glo annexin-V comparison to flow cytometry results




In this experiment, K562 cells were exposed to various doses of bortezomib for 16 hours.

Top Panel: Cells were harvested, washed, and labeled with Alexa Fluor® 488 (green fluorescence, PtdSer:AnxV binding) and 7-AAD (red fluorescence, membrane integrity) and analyzed by flow cytometry (10,000 events).

Bottom Panel: K562 cells (10,000/well) were exposed to serial dilutions of bortezomibin the presence of the RealTime-Glo™ Annexin  V Apoptosis Assay reagent. Luminescence (PtdSer:AnxV binding) and fluorescence (membrane integrity) were recorded kinetically. The 16 hour incubation with bortezomib is shown.


See More Data: Additional data illustrating how the real time method can be used to differentiate modes of cell death is available in the Poster A Real-Time, Plate -based Annexin V Method for Monitoring Programmed Cell Death, presented at AACR 2017.

Available With or Without Necrosis Detection Reagent

RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay
(Cat.# JA1011 and JA1012)

Includes Necrosis Detection Reagent, a cell-impermeant profluorescent DNA dye. Upon loss of membrane integrity, the dye enters the cell and binds to DNA, generating a fluorescent signal.

RealTime-Glo™ Annexin V Apoptosis Assay
(Cat.# JA1000 and JA1001)

Does not include Necrosis Detection Reagent. Although the assay can be conducted without a necrosis detection probe, the mechanism of action of PS exposure will not be revealed. Therefore, when using Cat.# JA1000 or JA1001, we recommend incorporating an asymmetric cyanine dye such as CellTox™ Green (Cat.# G8742) or structurally related molecules, for full assay functionality and data clarity. 

Specifications

You are viewing: JA1011 Change Configuration

What's in the box?

Item Part # Size

Annexin V-SmBiT Reagent (1,000X)

J700A 1 × 25μl

CaCl2 (1,000X)

J750A 1 × 250μl

Annexin V-LgBiT Reagent (1,000X)

J800A 1 × 25μl

Annexin V NanoBiT® Substrate, 1,000X

JA100A 1 × 25μl

Necrosis Detection Reagent, 1,000X

JA101A 1 × 25μl

SDS

Choose language:

Certificate of Analysis

Search for Specific Certificate:

View more results
No results
Loading…

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

Patents and Disclaimers

Patent Pending.

U.S. Pat. Nos. 8,598,198 and 9,458,499 and other patents and patents pending.

U.S. Pat. Nos. 9,797,889 and 9,797,890 and other patents pending.

Licensed under U.S. Pat. No. 6,949,350.

Specifications

You are viewing: JA1012 Change Configuration

What's in the box?

Item Part # Size

Annexin V-SmBiT Reagent (1,000X)

J700B 1 × 250μl

CaCl2 (1,000X)

J750A 1 × 250μl

Annexin V-LgBiT Reagent (1,000X)

J800B 1 × 250μl

Annexin V NanoBiT® Substrate, 1,000X

JA100B 1 × 250μl

Necrosis Detection Reagent, 1,000X

JA101B 1 × 250μl

SDS

Choose language:

Certificate of Analysis

Search for Specific Certificate:

View more results
No results
Loading…

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

Patents and Disclaimers

Patent Pending.

U.S. Pat. Nos. 8,598,198 and 9,458,499 and other patents and patents pending.

U.S. Pat. Nos. 9,797,889 and 9,797,890 and other patents pending.

Licensed under U.S. Pat. No. 6,949,350.

Specifications

You are viewing: JA1000 Change Configuration

What's in the box?

Item Part # Size

Annexin V-SmBiT Reagent (1,000X)

J700A 1 × 25μl

CaCl2 (1,000X)

J750A 1 × 250μl

Annexin V-LgBiT Reagent (1,000X)

J800A 1 × 25μl

Annexin V NanoBiT® Substrate, 1,000X

JA100A 1 × 25μl

SDS

Choose language:

Certificate of Analysis

Search for Specific Certificate:

View more results
No results
Loading…

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

Patents and Disclaimers

Patent Pending.

U.S. Pat. Nos. 9,797,889 and 9,797,890 and other patents pending.

Licensed under U.S. Pat. No. 6,949,350.

Specifications

You are viewing: JA1001 Change Configuration

What's in the box?

Item Part # Size

Annexin V-SmBiT Reagent (1,000X)

J700B 1 × 250μl

CaCl2 (1,000X)

J750A 1 × 250μl

Annexin V-LgBiT Reagent (1,000X)

J800B 1 × 250μl

Annexin V NanoBiT® Substrate, 1,000X

JA100B 1 × 250μl

SDS

Choose language:

Certificate of Analysis

Search for Specific Certificate:

View more results
No results
Loading…

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

Patents and Disclaimers

Patent Pending.

U.S. Pat. Nos. 9,797,889 and 9,797,890 and other patents pending.

Licensed under U.S. Pat. No. 6,949,350.

Choose your country

Americas

Brazil
Canada
United States

Pacific Asia

Australia
India
Japan
Korea, Republic of
Singapore

Europe

Austria
Belgium
Denmark
Estonia
Finland
France
Germany
Iceland
Italy
Luxembourg
Netherlands
Norway
Poland
Spain
Sweden
Switzerland
United Kingdom