A cell chip for sequential imaging of individual non-adherent live cells reveals transients and oscillations.

Advances in molecular cell biology, medical research, and drug development are driving a growing need for technologies that enable imaging the dynamics of molecular and physiological processes simultaneously in numerous non-adherent living cells. Here we describe a platform technology and software--the CKChip system--that enables continuous, fluorescence-based imaging of thousands of individual living cells, each held at a given position ("address") on the chip. The system allows for sequential monitoring, manipulation and kinetic analyses of the effects of drugs, biological response modifiers and gene expression in both adherent and non-adherent cells held on the chip. Here we present four specific applications that demonstrate the utility of the system including monitoring kinetics of reactive oxygen species generation, assessing the intracellular enzymatic activity, measuring calcium flux and the dynamics of target cell killing induced by conjugated cytotoxic T-lymphocytes. We found large variations among individual cells in the overall amplitude of their response to stimuli, as well as in kinetic parameters such as time of onset, initial rate and decay of the response, and frequency and amplitude of oscillations. These variations probably reflect the heterogeneity of even cloned cell populations that would have gone undetected in bulk cell measurements. We demonstrate the utility of the system in providing kinetic parameters of complex cellular processes such as Ca++ influx, transients and oscillations in numerous individual cells. The CKChip opens up new opportunities in cell-based research, in particular for acquiring fluorescence-based, kinetic data from multiple, individual non-adherent cells.

Simultaneous monitoring of binding to and activation of tumor-specific T lymphocytes by peptide-MHC.

The recent advent of peptide-MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide-MHC tetramers at the single-cell level. For this purpose, we have employed the CellScan, a non-flow cytometer designed for repetitive measurements of optical parameters (e.g., fluorescence intensity and polarization) of individual living cells. A melanoma-specific MART1 CTL line and a gp100-specific CTL clone were incubated with specific and control single-chain peptide-MHC tetramers for 45 min. Subsequently, the fluorescence intensity and polarization were measured by the CellScan. Specific binding of fluorescently labeled peptide-MHC tetramers to CTLs, recorded by the CellScan, was comparable to that measured by flow cytometry. CellScan monitoring of the degree of fluorescence polarization of fluorescein diacetate-labeled CTLs that were reacted with tetramers revealed specific activation of the CTLs, which was confirmed by cytokine (INF gamma) production. These results provide a new means of monitoring both the binding to and activation of T lymphocytes by cognate peptide-MHC complexes at the single-cell level, which can now be applied to distinguish between cognate responding and anergic T cells.

Tumor sensitivity to anti-cancer drugs predicted by changes in fluorescence intensity and polarization in vitro.

BACKGROUND: We used the CellScan, a novel static cytometer, to monitor changes induced by anti-neoplastic drugs in the fluorescence intensity and polarization of fluorescently-labeled tumor cells. MATERIALS AND METHODS: T47D and T80 human breast cancer cell lines were exposed to navelbine and to 5-fluorouracil and the fluorescence properties of the treated cells, stained with fluorescein diacetate and rhodamine 123, were measured by the CellScan. RESULTS: A strong correlation was found between the inhibition of cell growth induced by the two drugs, as estimated from cell counts, and the resulting changes in fluorescence intensity and polarization, as monitored by the CellScan. Fluorescence hyperpolarization of the labeled cells occurred in conjunction with AnnexinV binding and propidium iodide exclusion, indicating that such hyperpolarization, resulting from drug action, reflects an early stage of apoptosis, as previously proposed. CONCLUSION: The system presented here could serve as the basis for assessing drug sensitivity or resistance of cancer cells derived from small biopsies of solid human tumors, thus eliminating prior tumor culturing and time-consuming assays.