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Calculated CT+% was compared between nuclei from different regions of the spheroids, spheroid bottom, middle, and top (f) and spheroid outer cell layer and inner cell layers (g) For e-g, data is displayed for two independent experiments with two replicate plates per experiment. (e) Experimental validation compared the seeded CT+% of 500 cell T47D spheroids to the calculated CT+%, as determined by the segmentation analysis. Cyan outlines segmented nuclei and red outlines segmented nuclei determined to be CT+. (d) Images show segmentation examples for 50% and 3.125% CT spheroids, at 20 and 75 µm z-depths. The number of spheroids analyzed was 11, 25, 22, and 23 for 100%, 50%, 3.125%, and 0% seeded CT+ conditions, respectively. (c) Histogram depicts the CT intensity range in segmented nuclei, for control 0% CT spheroids, 100% CT+ spheroids, and 50% CT+ or 3.125% CT+. Color represents the average intensity of CT within the segmented nuclei. (b) Centroids of segmented nuclei within a 1 DIV 500 cell 50% or a 3.125% CT-labeled T47D spheroid. (a) Maximum intensity projection images of a 15 µm-thick region in the center of 1 DIV 500 cell T47D spheroids with nuclear Hoechst counterstaining and 50% or 3.125% CT labeling. (g) Heat map displays the clearing metric results, showing the average for each condition.Ĭell tracker titration experiment demonstrated utility of segmentation analysis in T47D spheroids. The number of spheroids analyzed for (b-g) can be found in Supplementary Table 2. Error bars represent SD, **P < 0.0001, *P < 0.05, ns = not significant. Clearing metric data is separated to see the effect of culture time on spheroids of a single size (e) and the effect of spheroid size on spheroids of a single age (f) For (e and f) two-way ANOVA with Tukey post-hoc multiple comparisons were performed. Data points represent individual spheroids, colors represent independent experiments, and horizontal bars represent averages for independent experiments.
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Approximate height (b), nuclear segmentation cutoff (c), and clearing metric (d) were calculated for three independent experiments for T47D spheroids at 3, 5, and 7 DIV and with 250, 500, 1,000 cells seeded per spheroid, for both cleared and uncleared conditions. Nuclear area was calculated by adding together the areas of each segmented nuclei within a slice. Spheroid slice area was calculated using the segmented whole spheroid. Calculation steps are demonstrated using example uncleared and cleared 3 DIV 250 cell spheroids. (a) Approximate spheroid height, nuclear segmentation cutoff, and clearing metric calculations. Ultimately this analysis pipeline allows for previously unattainable segmentation throughput of 3D culture models due to increased sample clarity and optimized batch-processing analysis.Īssessment of clearing and segmentation analysis in T47D spheroids. We demonstrate nuclear segmentation in multiple cell types, with accurate identification of fluorescently-labeled subpopulations, and develop a metric to assess the ability of clearing to improve nuclear segmentation deep within the tissue. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers.