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.