Real‐time cell viability assays using a new anthracycline derivative DRAQ7®

Abstract

The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a non-invasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be non-toxic to a panel of cancer cell lines grown continuously for up to 72 hours and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation and drug-induced cytotoxicity. The overall responses to anti-cancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multi-parameter and kinetic fingerprint of anti-cancer drug action.

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging

“…10 In this assay, Annexin V-positivity markedly preceded DRAQ7-positivity, which reflects the early exposure of PS followed by the unregulated loss of plasma membrane integrity during late stages of apoptosis (Supplementary Figure S1B). 11 In comparison, vehicle-treated cells cultured in the presence of Annexin V displayed no DRAQ7-positivity (Figure 1d) or changes in proliferation (data not shown) suggesting this method is non-toxic and well-tolerated.…”

Wrinkled, wavelength-tunable graphene-based surface topographies for directing cell alignment and morphology

“…As a model system, we chose to study murine and human fibroblasts on matched samples of 25 μm wavelength and flat s-GO substrates. Cell viability was measured on wrinkled and flat s-GO substrates compared with conventional tissue culture plastic (polystyrene) using DRAQ7 (Biostatus), a far-red fluorescent DNA dye that only stains dead and membrane compromised cells [55]. These measurements showed that cell viability was consistently over 95% on wrinkled and flat s-GO substrates (Supplementary Figure S7 and S8), indicating excellent biocompatibility.…”

Microflow Cytometers with Integrated Hydrodynamic Focusing

“…Flow cytometry has a number of applications, e.g., in microbiology and marine biology, but it is mostly applied in laboratory medicine, in particular for differentiation and counting of blood cells [8]. This type of application was also demonstrated in a number of microchips [1,3,5,9–15]. Numerous commercial solutions show that such technology could establish the basis for simple and robust analytical systems being of particular relevance for point-of-care in vitro diagnostics with a wide range of applications in emergency medicine and intensive care.…”

“…Fluid handling plays an important role for signal height and stability [14]. An essential part of the development of microflow cytometers is the control of the stream of micro particles in a flow channel [15]. Focusing of particles inside the flow channel significantly improves signal stability and prevents fouling of the channel walls.…”