title: Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry.
abstract: Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells.
authors.affiliation: The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Jin
authors.initials: J
authors.lastname: Akagi
authors.affiliation: The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Joanna
authors.initials: J
authors.lastname: Skommer
authors.affiliation: The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Anna
authors.initials: A
authors.lastname: Matuszek
authors.affiliation: R&D Division, On-chip Biotechnologies Co. Ltd, Tokyo, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takeda
authors.affiliation: R&D Division, On-chip Biotechnologies Co. Ltd, Tokyo, Japan.
authors.forename: Yuu
authors.initials: Y
authors.lastname: Fujimura
authors.affiliation: School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia.
authors.forename: Khashayar
authors.initials: K
authors.lastname: Khoshmanesh
authors.affiliation: School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia.
authors.forename: Kourosh
authors.initials: K
authors.lastname: Kalantar-Zadeh
authors.affiliation: School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia.
authors.forename: Arnan
authors.initials: A
authors.lastname: Mitchell
authors.affiliation: School of Medicine, Cardiff University, Cardiff, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Errington
authors.affiliation: School of Medicine, Cardiff University, Cardiff, UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Smith
authors.affiliation: The Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, NY, USA.
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.affiliation: The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand ; School of Applied Sciences, RMIT University, Melbourne, Australia.
authors.forename: Donald
authors.initials: D
authors.lastname: Wlodkowic
keywords: Lab-on-a-Chip; apoptosis; cancer; cell cycle; flow cytometry; microfluidics; programmed cell death