![]() ![]() Measurements of internal spheroid structure provide the most insight, whereas varying initial spheroid size and temporal measurement frequency is less important. However, using an objective mathematical modelling framework and statistical identifiability analysis we quantitatively compare experimental designs and identify design choices that produce reliable biological insight. These data are difficult to compare and interpret. By intentionally varying the initial spheroid size and temporal sampling frequencies across multiple cell lines, we collect an abundance of measurements of internal spheroid structure. Using multiple experimental designs, live-dead cell staining, and real-time cell cycle imaging, we measure necrotic and proliferation-inhibited regions in over 1000 4D tumour spheroids (3D space plus cell cycle status). ![]() Various and inconsistent experimental designs are used, leading to challenges in interpretation and reproducibility. Tumour spheroid experiments are routinely used to study cancer progression and treatment. The present article elaborates how the method yields high-quality two-and 3D images that provide information about inter-cell variability and inner spheroid structure. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. ![]()
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