Live/Dead Staining and Quantitative Analysis

Treatment efficacy was determined by in situ Live/Dead staining using calcein AM (ThermoFisher) and propidium iodide (Sigma–Aldrich), followed by fluorescence imaging (Olympus FV1000 confocal laser scanning microscope), as optimized for 3D cultures [26 (link)]. Derivation of outcome parameters was done using quantitative image analysis according to the CALYPSO methodology, as described previously [27 (link)]. When indicated, culture viability was assessed based on NAD(P)H oxidase activity using a CellTiter 96 aqueous non-radioactive proliferation assay (MTS, Promega, Madison, WI, USA), which was performed in accordance with the manufacturer’s recommendation.

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Broekgaarden M., Alkhateeb A., Bano S., Bulin A.L., Obaid G., Rizvi I, & Hasan T. (2020). Cabozantinib Inhibits Photodynamic Therapy-Induced Auto- and Paracrine MET Signaling in Heterotypic Pancreatic Microtumors. Cancers, 12(6), 1401.

Publication 2020

calcein AM propidium iodide FV1000 confocal laser scanning microscope CellTiter 96 aqueous non-radioactive proliferation assay

Assay Calcein Calypso Confocal laser scanning microscope Nad p h oxidase Promega Propidium iodide Radioactive

Corresponding Organization : Harvard University

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Variable analysis

independent variables

Treatment

dependent variables

Cell viability (based on Live/Dead staining and fluorescence imaging)
Cell viability (based on NAD(P)H oxidase activity using MTS assay)

control variables

3D cell culture conditions (as optimized in previous work [26])
CALYPSO methodology for quantitative image analysis (as described in previous work [27])

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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