Apoptosis Assays for High-Throughput Cytometry  

Apoptosis is an essential process for normal tissue development and homeostasis by which cells undergo timely programmed cell death. Aberrations in apoptotic signaling are implicated in a range of human pathologies including cancer, autoimmune disease and neurodegeneration. Induction of apoptosis leads, in most cases, to the depolarization of mitochondria, the activation of caspases and plasma membrane alterations. The ability to simultaneously measure a multitude of apoptotic markers allows confirmation of pathway and insight into mechanism of action over time.


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Assay Concept

Introducing Apoptosis Assays for iQue® High-Throughput Screening (HTS)...

Figure 1. Apoptotic markers measured by the iQue® Human 4-Plex Apoptosis Kit. 1) Mitochondrial depolarization, 2) Caspase 3/7, 3) Annexin V and 4) Non-viable. The kit components can be utilized either separately or combination in a no wash format. Apoptosis reagents can also be multiplexed with other iQue® HTS reagents in no wash assays.

Key Advantages

Key Advantages of Apoptosis Assays

  • Detect and confirm apoptosis through different pathways using the mix and match reagents
  • Gain a robust determination of apoptosis progression and insight into mechanism of action
  • Simple mix-and-read 96 and 384-well protocol with no washing and no fixing

Detect and Confirm Apoptosis Through Different Pathways Using the Mix and Match Reagents

Figure 2. Cytotoxic and cytostatic compounds produce different cell health profiles. Jurkats (1e6/mL) treated with a range of concentrations of 4 compounds for 24 hours. Before analysis with iQue®️ Human 4-Plex Apoptosis Kit.

(A) Caspase 3/7 expressing cells are shown in a heatmap from iQue Forecyt®️.

(B,C,D,E) Depolarized mitochondria, Caspase 3/7, Annexin V and Non-viable (respectively) cell percentages are shown.

Gain a Robust Determination of Apoptosis Progression and Insight Into Mechanism of Action

Figure 3. Cell health markers show a time dependent increase when treated with an apoptosis inducing drug.
Jurkats (1e6/mL) treated with a range of concentrations of Staurosporin. 20 µL samples were taken and analyzed after 2, 6 and 24 hours of treatment with 3 apoptosis reagents. (A) Heat map of Caspase positive cell (%) from 2 hours, (B) Mitochondrial depolarization, (C) Caspase and (D) Annexin V

Simple Mix-and-Read 96 and 384-Well Protocol With No Washing and No Fixing

Figure 4. Schematic of the simple, mix and read apoptosis assay.

Ordering Information

Ordering Information

iQue®️ Human 4-Plex Apoptosis Kit
Platform: Compatible with iQue®️ 3 BR/VBR & iQue®️ Plus BR/VBR
Available SizesCatalog Numbers
1 x 38490053

 

Individual Apoptosis Reagents
Platform: Compatible with iQue®️ 3 BR/VBR & iQue®️ Plus BR/VBR
ProductDetection ChannelAvailable SizeCatalog Number
iQue® Human Caspase 3/7B/Green5 x 38491034
iQue® Human Annexin VB/Yellow5 x 38491030
iQue® Mitochondrial Membrane PotentialR/Red5 x 38491038
iQue® Cell Membrane IntegrityB/Red5 x 38490346

 

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Resources

Literature and Documentation

Application Note

Comparison of Anti-TNFα Monoclonal Antibody Biosimilars

Combining Binding and Functional Analyses for Comparison, of Anti-TNFα Monoclonal Antibody Biosimilars

Illustration of the destruction of a human cell by lysis- shows apoptosis and mitochrondria

Exploiting the Mitochondrial Pathway of Apoptosis

Understanding the intrinsic, or mitochondrial, pathway of apoptosis has produced potent targeted therapies such as venetoclax.

iQue®️ Reagent Kits brochure cover
Brochure

iQue® Reagent Kits

Perform comprehensive, multiplexed analysis of cells, beads and secreted proteins all from the same cells at the same timepoint.

Brochure

iQue® High-Throughput Screening (HTS) Cytometry Platform Brochure

Product Guide

 iQue®️ Apoptosis Kits

 iQue®️ Human 4-Plex Apoptosis Kit

Infographic

A cytometry approach to assessing cell health and viability

Application Note

iQue® Human 4-Plex Apoptosis Kit Using Cytometry

A robust assay for measuring cellular progression through apoptosis via multiple pathways.

Related Applications

Application Note

Development of a no-wash 4-plex apoptosis screening kit and validation for phenotypic screening

Cell Count and Viability

Information can be used by monitoring cell proliferation rates, optimizing growth conditions and normalizing cell data for further studies.

Fixable Viability Dyes

Determining cell viability is crucial when assessing a cell’s response to treatment in order to exclude them from final data analysis.

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