Cytotoxicity testing and screening with high-throughput flow-cytometry

Cytotoxicity testing via flow cytometry is a fundamental assessment at any step of the drug discovery journey, from early investigations to late safety evaluations. Exposure of cells to a cytotoxic stimulus can compromise membrane integrity, impair metabolic activity, inhibit cell proliferation, or ultimately lead to cell death. In clinical trials, potential drug candidates often fail because of unexpected toxicity. Early cytotoxicity tests provide a crucial means to rank compounds for further consideration: While a compound inducing a cytotoxic response may be eliminated from subsequent screening rounds, a compound with anti-proliferative activity may represent a potential candidate for cancer treatment.

Common methods to screen cell viability are based on the determination of the cell membrane’s integrity. This is simply done by using fluorescent dyes, which can penetrate the membranes of damaged/dying cells and bind to nucleic acids inside the cell nucleus. Propidium iodide (PI) dyes are widely employed to measure viability by flow cytometry due to their low cost, rapid cell penetration kinetics, convenient excitation, and emission spectra. However, their applicability to high-throughput viability screening is challenged by the inherent nature of PI to penetrate the membranes of healthy cells after prolonged exposure times. 

Features of different PI staining procedures

MACSQuant® Flow Cytometers (MACSQuant Analyzer 10, MACSQuant VYB, and MACSQuant X) overcome the limitations of PI assays. With integrated robotic pipetting arms, they automatically pipette fresh PI directly into each sample and standardize the incubation and labeling process. This, together with the instrument’s automated sampling of 96-well plates, provides a robust, automated, hands-free, and high-throughput setup for all cell viability experiments.

Application note
Reliable, hands-free, and high-throughput cell viability assays on the MACSQuant® Flow Cytometer

Apoptosis, or programmed cell death, is one way that cells can react to an external stimulus. Several different apoptotic pathways are well described, including both extrinsic and intrinsic drug-induced pathways. A non-inflammatory apoptotic response differs from necrosis (a more pro-inflammatory type of cell death), which could be highly damaging if induced by a drug. Therefore, cytotoxicity screening is essential during preclinical drug safety testing.

Gating scheme for analysis of flow cytometric data

In this study by Cayman Chemical, a high-throughput multiparameter apoptosis screening platform was set up. By combining the Early Apoptosis Detection Assay Kit (Cayman) with the MACSQuant X flow cytometer, distinct stages of apoptosis could be quantitively distinguished within a cell population. The results showed that multiple cellular parameters can be analyzed in a single experiment in a highly automated manner, increasing the reproducibility and sensitivity of the cytotoxicity screening.

Application note
High-throughput flow cytometric screening of a kinase inhibitor library for apoptosis induction

Julie M. Rumble and David L. Hoffman
Bioanalysis & Assay Development Services, Cayman Chemical Company