When you input the appropriate cell size, background intensity, and Z/XY ratio (Z-axis elongation of the nucleus), NoviSight software can automatically detect the spheroid’s nuclei in 3D space (Figure 3A).įor all the nuclei stained with Hoechst 33342, the ones with a high NucView 550 signal intensity were dead cells. To recognize the cell nucleus, we used a specialized NoviSight module for nuclear detection: NuclearL. NoviSight software can detect the number of cells from the nuclear stains. The live/dead cell assay was evaluated by the number of live/dead cells. Table 1: 3D analysis workflow from sample preparation to analysis. confocal microscopy, multiphoton microscopyġ. If you want to analyze cell number, you should stain the nucleusĮx. In order for NoviSight software to three dimensionally recognize the nuclei from the Z-plane images, set the step size of the Z-section at 3 μm-the largest measurement to detect a single nucleus. The steps outlined in Table 1 are required to quantitatively analyze live/dead spheroids using NoviSight™ software. Intuitive user interface helps you transform Z-stack images into reproducible quantitative data.Easily perform a 3D live/dead viability test.Overall, STS induced the cell death of the HT-29 spheroids.įigure 2: Volumetric view of HT-29 spheroids with (right) and without (left) STS. The core of a non-treated, 8-day cultured spheroid was dead likely due to low nutrition or hypoxia. Figure 2 shows non-treated and STS-treated spheroids’ volumetric images acquired using the microscope. A 405 nm laser was used for Hoechst 33342 (blue fluorescence), and a 561 nm laser was used for NucView 550 (magenta fluorescence). You can see the acquisition conditions below. We imaged the samples in three dimensions using the FV3000 confocal microscope and a semi-apochromat bjective lens (LUCPLFLN20X). After staining, the spheroids were cleared with SCALEVIEW-S4. The spheroids’ nuclei were stained with Hoechst 33342 at 4 ☌ (39.2 ☏) overnight.
After treatment with STS and NucView 550, the HT-29 spheroids were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100/PBS(-). NucView 550 is a caspase-3 fluorescent probe, and the dead cell nuclei were fluorescently stained with red. We cultured 500 HT-29 cells/well in a PrimeSurface® 96U plate (Sumitomo Bakelite) for eight days to form spheroids, then applied various concentrations of staurosporin (STS) with NucView® 550 (Biotium) the next day.
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In this application note, we demonstrate how to quantitatively analyze 3D samples with NoviSight software using a live/dead spheroids assay.įigure 1 (left to right): 2D, 2.5D, and 3D analysis schematic. Both techniques lose a lot of data from the 3D sample, such as spatial, morphological, and volumetric information (Figure 1). On the other hand, conventional analysis uses a single image slice (Figure 1, left, 2D analysis) or uses a projection image (Figure 1, center, 2.5D analysis). NoviSight software’s true 3D analysis can simplify this process by measuring volume, the number of cells, and the cell condition of 3D samples based on sequential Z-stack fluorescent images. Ideally, we can analyze 3D samples three dimensionally to obtain information on their morphology and interior condition. This application note will introduce the NoviSight 3D cell analysis workflow for a live/dead spheroids assay.ģD high-content analysis (HCA) using spheroids or organoids can be difficult. There are eight colors of amine-reactive dyes to choose from, offering flexibility in experimental design.NoviSight 3D cell analysis software provides statistical data on spheroids and other 3D objects by measuring parameters like volume, sphericity, and cell numbers. The reactive dye can enter dead cells and label proteins in the interior of the cell, producing at least a 50-fold increase in fluorescence.īecause the LIVE/DEAD® dyes react covalently with proteins, dead cell discrimination is completely preserved following fixation of the sample with formaldehyde under conditions that are commonly used for intracellular immunophenotyping and to inactivate pathogens. In live cells, only surface proteins bind to the reactive dye, resulting in dim fluorescence. Often referred to as amine-reactive dyes, these stains are based on the reaction of a fluorescent reactive dye with cellular proteins. However, the LIVE/DEAD® fixable kits are designed to assess cell viability in samples after fixation and/or permeabilization (Figure 4). Like the SYTOX® dyes, the LIVE/DEAD® Fixable Dead Cell Stain Kits utilize the loss of membrane integrity for dead cell discrimination (Figure 3).
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