Cell Biology Research and Development

Figure 1. The Via2-Cassette™ preloaded with Acridine Orange and DAPI. The volume calibrated single use Via2-Cassette™ is a true all-in-one solution for optimized cell count. The cassette is a disposable plastic unit that contains the fluorescent dye used for analysis. A cell sample is easily loaded into the cassette by submerging the inbuilt pipette into the cell suspension and pressing the piston.

Manual cell counting using a hemocytometer and the trypan blue exclusion method is time consuming and depends heavily on the subjective perception of the user. For the automated cell count and viability determination in the NucleoCounter^® instruments NC-202™ and NC-3000™, all you need to do is to load the cell suspension into the Via2-Cassette™ (Figure 1). Cells are automatically stained by the two fluorophores Acridine Orange and DAPI staining total cells and dead cells, respectively. The pre-calibrated volume of analysis chamber ensures high precision and reproducibility. The image cytometry with the NucleoCounter^® instruments NC-202™ and NC-3000™ allows for detailed data analysis and fast acquisition of results.

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Figure 2. Using the A8-Slides™ allows for fast viability and cell count with 8 samples in less than 3 minutes. The cell suspension is previously mixed with the fluorophores Acridine Orange and DAPI to stain the total cell population and the dead cells, respectively.

The multi-chamber A8-slide™ (Figure 2) enables high speed viability and cell count determinations of insect and mammalian cells with 8 counts in less than 3 minutes. Simply mix cell suspension with the fluorescent dyes acridine orange and DAPI to stain the total cell population and the dead cells, respectively. Then, the chamber is loaded and the A8-slide™ inserted into the NucleoCounter^® instruments NC-250™ and NC-3000™. ChemoMetec A/S even offers optimized counting protocols for aggregated cells, cells growing on microcarriers and in spheroids.

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Figure 3. Inspection of the counted cells within the Nuceloview™ software. To verify the counting, cells can be tracked by visual inspection of the plotted data from the data plot towards the image and from the image towards the data plot.

When counting manually all events can be visually identified and verified. The NucleoView™ software also allows visual inspection of the fluorescence image and the opportunity to verify the counting. Specific event populations can be selected in the scatter plots and examined visually to determine the validity of their inclusion or exclusion from the final counting results. The adapted protocol can be saved for further measurements.

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Figure 4. Schematic representation of the “Viability and Cell Count – Aggregated Cells Assay” protocol. Even aggregating cells and samples with large cell clumps can be counted with the NucleoCounter^® Aggregated Cells Assay. In the aggregated cells assay, the total cell count derives from a lyzed aliquot of the sample (see schematic representation above). Lysis disrupts the cell membrane, dismantles clumps and releases nuclei into suspension. Nuclei are stained by DAPI and automatically counted. Because dead cells are typically less abundant and tend to detach from aggregates, the dead cell count can be determined from the non-lyzed sample (above figure). The combination of these two measurements results in high precision and accuracy even in heavily clumpy samples.

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For advanced research in cell death it is essential to study the mechanisms of apoptosis. A series of 5 plug-and-play assays in the NucleoCounter^® NC-3000™ 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 (Table 1). Covering early to late stages of apoptosis, these assays will allow for detailed study of progress of apoptosis in mammalian cells. With the user friendly accompanying Nuceloview™ software, advanced data analysis can effortlessly be performed. The unique possibility to link between images, histograms and scatter plots allows for detailed and precise data analysis identifying percentages of apoptotic, necrotic and living cells.

Cells are often transfected with genes of interest. One method to follow the expression of these genes is to co-express a fluorescent protein such as green fluorescent protein (GFP) under the same promoter and determine the amount of GFP. ChemoMetec A/S offers a fast and easy assay for testing GFP transfection with the NucleoCounter^® NC-3000™ (Figure 4). 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 5). The NucleoCounter^® instrument NC-3000™ offers an all-in-one platform for evaluating GFP expression efficiency.

Figure 5. Principle of the plug-n-play GFP transfection efficiency assay using the NucleoCounter^® NC-3000™.

• (A) Cells are located using Hoechst 33342 (blue) and the percentage of GFP expressing cells (green) can easily be determined. Nonviable cell are stained with propidium iodide (PI; red).
• (B) In the accompanying NucleoView™ software, all cells stained with Hoechst 33342 (blue) are identified.
• (C) GFP expressing cells are identified in green and inviable cells in red by PI staining.

 

Figure 6. Coupling between the obtained image and the scatter plots allows for visual inspection of the precision of defined gates.

• (A) With the image overlay function in the NucleoView™ software, the non-viable cells stained with propidium iodide (PI) can be located.
• (B) The GFP-transfected cells can be easily identified.

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Figure 7. 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 cell biology. The NucleoCounter^® family instruments NC-250™ and NC-3000™ provide fast and easy cell cycle analysis in less than 5 minutes (Figure 7). 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 7. Possible LED and emission filter combinations in the FlexiCyte™ module for the NucleoCounter^® NC-3000™. This allows for detection of a broad range of fluorescent antibodies and proteins and for the detailed analysis of biomarkers.

The FlexiCyte™ module in the NucleoCounter^® NC-3000™ enables users to perform detailed advanced cell analysis of a broad range of biomarkers and fluorescent proteins in mammalian cells (Figure 7). The combination of LEDs from UV to far red with a carefully selected set of emission filters allows for detection of a range of fluorescent antibodies and proteins. The ‘Protocol Adaptation Wizard’ guides the user through selection of optimal settings. After image acquisition, cell data are presented beside the fluorescent image as either scatter plots, histograms or both in the plot manager. Detailed data analysis can then be performed by linkage between images and plots.

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