Applications in Toxicology

Figure 1. Fast screening of cytotoxicity with the NucleoCounter^® instruments NC-250™ and NC-3000™. In less than 3 minutes, 8 viability and cell counts can be performed.

• (A) All cells are stained with Acridine Orange (green).
• (B) Dead cells with an impaired cell membrane will be additional stained with DAPI (blue).

When assaying the effect of a potential toxic compound, it is essential to precisely evaluate the viability of a cell population through assessment of membrane integrity. The fluorescent stain DAPI will only penetrate cells with an impaired plasma membrane and acridine orange will stain the total cell population. Combined with the NucleoCounter^® instruments NC-250™ and NC-3000™, counting and viability determinations of mammalian cells have never been more uncomplicated. Simply mix a small volume of the cell suspension with fluorescent dyes, load and run. Within 3 minutes you will have 8 high precision viability and cell count determinations allowing for fast screening of cytotoxicity.

Viability and Cell Count - Mammalian Cells .pdf

In order to study cytotoxicity it is required to determine the viability of cell populations. Cells can undergo necrosis where membrane integrity is lost and where cells rapidly will die due to cell lysis. Cytotoxicity might also trigger apoptosis, where cells will die by genetically programmed cell death with a series of hallmarks. It is therefore of fundamental importance to study the detailed mechanism of cell death in both treated and untreated cells. Determining whether cell death is caused by necrosis or apoptosis, whether cells are early- or late-apoptotic and whether it is the intrinsic or extrinsic pathway of apoptosis is easy using the NucleoCounter^® instrument NC-3000™. A series of 5 plug-and-play assays allow for full investigation of the cell death mechanism including Annexin V, mitochondrial potential with JC-1, caspase signaling, DNA fragmentation and the unique 1-minute vitality assay covering early to late apoptosis (Table 1).

Figure 2. Fast cell cycle analysis.

• (A) The different cell cycle phases can be distinguished based on the DNA content using a fluorescent DNA stain.
• (B) Two-step cell cycle assay of untreated and camptothecin-treated (CPT) Jurkat cells. The histograms display intensity of the DNA-stain DAPI and can be used to define cell cycle events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase. After CPT treatment, the cell cycle is arrested in the G2/M-phase.

Investigating the impact of a treatment on cell division is one of the most powerful tools within cytotoxicity. The NucleoCounter^® instruments NC-250™ and NC-3000™ provide fast and easy cell cycle analysis in less than 5 minutes (Figure 2). After addition of a lysis buffer, all cell nuclei are stained and the sample can be measured using the NucleoCounter^® instruments. A cell cycle profile will readily be displayed in the accompanying NucleoView™ software and events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase can be identified. With the FlexiCyte™ software package, the NucleoCounter^® NC-3000™ can even be used for studying cell proliferation with e.g. BrdU und EdU incorporation detected with fluorescently labeled antibodies allowing for advanced studies in cell proliferation.

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Figure 3. Principle of the plug-n-play GFP transfection efficiency assay using the NucleoCounter^® NC-3000™.

• (A) Cells are located using Hoechst 33342 and are visible in blue.
• (B) The GFP expressing cells are visible in green.
• (C) Nonviable cells are stained with propidium iodide (PI; red).
• (D) All channels can be viewed as overlay.
• (E) Detailed data analysis can be performed with the accompanying NucleoView™ software.

Green fluorescent protein (GFP) is frequently used as a marker for transfection, but transfection with certain genes can be toxic for cells. It is therefore extremely important to determine the effect of transfection on cell viability while assaying the transfection rate. ChemoMetec A/S offers a fast and easy assay for testing GFP transfection with the NucleoCounter^® NC-3000™ (Figure 3). Cells are stained with Hoechst 33342 and propidium iodide (PI) to define the total cell population and the dead cell population together with the population expressing GFP. Easy coupling between the image obtained and the scatter plots or histograms allows for very precise determination of the gate settings used for counting of populations (Figure 3). The NucleoCounter^® instrument NC-3000™ offers an all-in-one platform for evaluating GFP expression efficiency. With the user-adaptable module FlexiCyte™, transfection percentage of cells expressing e.g. red fluorescent protein (RFP) or yellow fluorescent protein (YFP) can also easily be determined.

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