High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity

Part of the Methods in Molecular Biology book series (MIMB, volume 1601)


Mainstream adoption of physiologically relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price, and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily available reagents and open-source software to analyze spheroid volume, metabolism, and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination—for both single images and images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyze plate uniformity (such as edge effects and systematic seeding errors), detect outliers, and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.

Key words

Alamar blue Viability assays Overlay culture Hanging drop FiJi ImageJ Image analysis Three-dimensional cell culture In vitro model Preclinical screening Drug sensitivity 



Delyan Ivanov was supported by an EPSRC Doctoral Prize award hosted by the University of Nottingham (DP2014/DI). The authors would like to thank Pamela Collier and Alan McIntyre for their help with manuscript editing. Special thanks to Neli Garbuzanova, Janhavi Apte, Arundhati Dongre, Parminder Dhesi, and Amarnath Pal for testing the macros and providing user feedback.

Supplementary Files

The macro files and Excel spreadsheet are available through the Figshare database:

Macro 1 link: (DOI:  10.6084/m9.figshare.3487919).

Macro 2 link: (DOI:  10.6084/m9.figshare.3487943).

Volume analysis template: (DOI:  10.6084/m9.figshare.3487940).


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, Queen’s Medical CentreUniversity of NottinghamNottinghamUK
  2. 2.School of PharmacyUniversity of NottinghamNottinghamUK

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