Modernizing the MTT assay with microfluidic technology and image cytometry

Abstract

THE combination of microfluidic technology and imaging provides a new set of cytometric tools that can be quantitative and highly informative. Microfluidic devices can be used to capture small numbers of cells and expose those cells to a unique set of conditions (e.g., growth factors, toxins, and shear forces) by controlling the chemical and physical environment around the cells. The processes used to capture cells and manipulate the environment can all be controlled ‘‘on-chip,’’ reducing the number of manual handling steps, allowing for automation, minimizing the quantities of reagents required. Because the microfluidic device can be frequently placed on a microscope stage, quantitative, time lapse imaging can be used to record the dynamic responses of the cells. In the extreme, large numbers of individual cells can be specifically manipulated all while images are acquired on an automated microscope. In this issue of Cytometry, Lim et al. (page 691) adapt the standard methyl tetrazolium (MTT) assay (1) to a microfluidic device and use automated imaging and image analysis to obtain single cell measurements. The MTT assay is a colorimetric assay that is typically performed in 96-well microtiter plates and at the end of the assay an absorbance measurement is made from each well using a plate reader. In routine applications, the MTT assay is used to assess cell viability or to measure cell proliferation. Because pharmaceutical agents or toxins can stimulate or inhibit cell growth, the MTT assay is frequently used as cytotoxicity assay. Typically, the treated and control cells are incubated for 2–4 h with the MTT reagent, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, at concentrations in the range of 1 mM. Metabolically active cells reduce the MTT reagent into purple formazan crystals, presumably through mitochondrial enzymes such as succinate dehydrogenase. Before reading the plate, the crystals are dissolved with sodium dodecyl sulfate, and then the absorbance is measured for each well. The results of the standard MTT assay are ambiguous because the absorbance measurement alone cannot be used to distinguish between changes in average enzyme activity of the cell population from changes in the number of cells that can occur by either cell proliferation or death. Furthermore, the