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DOI: 10.1369/jhc.4A6301.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (1): 23-34, 2005
Copyright ©The Histochemical Society, Inc.

Quantification of Viability in Organotypic Multicellular Spheroids of Human Malignant Glioma using Lactate Dehydrogenase Activity : A Rapid and Reliable Automated Assay

Philip C. De Witt Hamer, Ard Jonker, Sieger Leenstra, Jan M. Ruijter and Cornelis J.F. Van Noorden

Departments of Neurosurgery (PCDWH,SL), Cell Biology and Histology (AJ,CJFVN), and Anatomy and Embryology (JMR), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands

Correspondence to: Philip C. De Witt Hamer, MD, Academic Medical Centre, University of Amsterdam, Dept. of Neurosurgery, Room H2-230, PO Box 22660, 1100 DD Amsterdam, The Netherlands. E-mail: P.C.deWittHamer{at}amc.nl

Organotypic spheroids from malignant glioma resemble the biological complexity of the original tumor and are therefore appealing to study anticancer drug responses. Accurate and reproducible quantification of response effect has been lacking to determine drug responses in this three-dimensional tumor model. Lactate dehydrogenase (LDH) activity was demonstrated in cryostat sections of spheroids using the tetrazolium salt method. Calibrated digital image acquisition of the stained cryostat sections enables quantification of LDH activity. Fully automated image cytometry reliably demarcates LDH-active and LDH-inactive tissue areas by thresholding at specific absorbance values. The viability index (VI) was calculated as ratio of LDH-active areas and total spheroid tissue areas. Duplicate staining and processing on the same tissue showed good correlation and therefore reproducibility. Sodium azide incubation of spheroids induced reduction in VI to almost zero. We conclude that quantification of viability in cryostat sections of organotypic multicellular spheroids from malignant glioma can be performed reliably and reproducibly with this approach. (J Histochem Cytochem 53:23–34, 2005)

Key Words: spheroids • glioma • lactate dehydrogenase • enzyme histochemistry • metabolic activity • toxicity test • biological assay • drug screening assays • image cytometry • cryostat sections


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