Dimension of apoptotic markers in tumors could be correlated with the cell routine stage using stream cytometry (FCM) directly. of the portable and user-friendly microfluidic stream cytometer (μFCM) that may perform multivariate real-time evaluation on Mouse monoclonal to THAP11 live cells using sampling amounts no more than 10 microliters. The μFCM program employs throw-away microfluidic cartridges fabricated using shot molding in poly(methylmethacrylate) clear thermoplastic. Furthermore the devoted and miniaturized digital hardware interface allows up to six parameter recognition using a mix of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for speedy laser alignment modification. We provide brand-new evidence a simple 2D circulation 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 any dose-response profiling of investigational anti-cancer brokers on human hematopoietic malignancy cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to standard 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. Keywords: microfluidics Lab-on-a-Chip circulation cytometry cell cycle apoptosis programmed cell death malignancy 1 INTRODUCTION Considerable progress is currently being made Decernotinib in our understanding of a diversity of existing modes of programmed tumor cell death.1-3 The cell propensity to undergo so called classical apoptosis is a key mechanism in the pathogenesis of many human diseases.1-3 The term apoptosis is used here to define the “classical” apoptotic process initially also called programmed cell death (PCD) which is inclusive of such hallmarks as: (i) mitochondrial changes manifesting by collapse of the transmembrane potential and release of cytochrome c to cytosol (ii) activation of caspases (iii) chromatin condensation (pycnosis) (iv) activation of endonuclease(s) followed by internucleosomal DNA cleavage (v) segregation of nucleoli (vi) fragmentation of nucleus and (vii) plasma membrane blebbing associated with formation of apoptotic bodies.4 5 In the context of raising clinical relevance of tumor cell death in malignancy therapy there is an ever increasing demand for convenient analytical tools to rapidly quantify and characterize apoptotic cell death.4-7 High-content analysis is widely recognized as a key component in the anti-cancer drug discovery pipelines. Multiparameter circulation cytometry (FCM) represents one of the most important techniques for quick quantification of programmed cell death.4 8 Vast majority of classical attributes of apoptosis can be quantitatively examined by flow cytometry.4-6 It allows assessment of multiple cellular characteristics on a single cell level. The possibility of quick measurement of individual cells in large Decernotinib cell populations offered by circulation cytometry Decernotinib made it the methodology of choice to identify and quantify apoptotic cells as well as to study mechanisms associated with this mode of cell death.4-5 By virtue of multiparameter analysis cytometry allows correlative studies between many cell attributes based on both light scatter and fluorescence measurements.4-5 An example of this is that measurements of apoptotic markers in tumors can be directly correlated with the DNA content (cell cycle phase) using FCM.12-13 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.4 5 The access to Decernotinib FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation.4-6 This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic circulation cytometer (μFCM) that can perform multivariate analysis on live tumor cells using minimal sampling volumes. We provide new evidence that a simple 2D circulation 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 any dose-response profiling of investigational anti-cancer.