Protocol for the generation and automated confocal imaging of whole multi-cellular tumor spheroids

Benjamin Genenger; Luke McAlary; Jay R Perry; Bruce Ashford; Marie Ranson

doi:10.1016/j.xpro.2023.102331

Protocol for the generation and automated confocal imaging of whole multi-cellular tumor spheroids

STAR Protoc. 2023 Jun 9;4(2):102331. doi: 10.1016/j.xpro.2023.102331. Online ahead of print.

Authors

Benjamin Genenger¹, Luke McAlary², Jay R Perry², Bruce Ashford³, Marie Ranson⁴

Affiliations

¹ School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia. Electronic address: bg038@uowmail.edu.au.
² School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia.
³ Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia; School of Medicine, University of Wollongong, Wollongong, NSW, Australia.
⁴ School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia. Electronic address: mranson@uow.edu.au.

Abstract

Multi-cellular tumor spheroids (MCTS) have found widespread use in pre-clinical research. However, their complex three-dimensional structure makes immunofluorescent staining and imaging challenging. Here, we present a protocol for whole spheroid staining and automated imaging using laser-scanning confocal microscopy. We describe steps for cell culture, seeding of spheroids and transfer of MCTS, and adhesion to Ibidi chamber slides. We then detail fixation, immunofluorescent staining based on optimized reagent concentrations and incubation times, and confocal imaging facilitated by glycerol-based optical clearing.

Keywords: Cancer; Microscopy.