Apoptosis Assays for Advanced High Throughput Flow Cytometry

Apoptosis Pathway

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

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® reagents in no wash assays.

Key Advantages

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

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

Ordering Information

Technical Resources

Documents

iQue®️ Human 4-Plex Apoptosis Kit Product Guide

Application Notes

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

Evaluating cellular progression through apoptosis via multiple different pathways

Posters

ELRIG 2014: High throughput multiplexed apoptosis assays using the Labcyte Echo Liquid Handler and the Intellicyt iQue Screener HD

SLAS 2014: Rapid profiling of compounds for cell cycle and apoptosis: using single micro-scale compound treatments for multiple multiplexed assays

Infographic

An advanced flow cytometry approach to assessing cell health and viability