Summary We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This non-invasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested by screening a library containing FDA approved drugs, experimental compounds and nature compounds. Compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRP). The compounds were clustered based on TCRP similarity. We identified novel mechanisms for existing drugs, confirmed previously reported calcium modulating activity for COX-2 inhibitor, celecoxib and identified an additional mechanism for the experimental compound, monastrol. We also identified and characterized a new anti-mitotic agent. Our findings indicate TCRP approach provides predictive mechanistic information for small molecule compounds.

A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 101 to 106 plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT50 or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT50 was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies.

Cellular processes such as cell cycle progression, mitosis, apoptosis, and cell migration are characterized by well-defined events that are modulated as a function of time. Measuring these events in the context of time and its perturbation by small molecule compounds and RNAi can provide mechanistic information about cellular pathways being affected. We have used impedance-based time-dependent cell response profiling (TCRP) to measure and characterize cellular responses to antimitotic compounds or siRNAs. Our findings indicate that small molecule perturbation of mitosis leads to unique TCRP. We have further used this unique TCRP signature to screen 119 595 compound library and identified novel antimitotic compounds based on clustering analysis of the TCRPs. Importantly, 113 of the 117 hit compounds in the TCRP antimitotic cluster were confirmed as antimitotic based on independent assays, thus establishing the robust predictive nature of this profiling approach. In addition, potent and novel agents that induce mitotic arrest either by directly interfering with tubulin polymerization or by other mechanisms were identified. The TCRP approach allows for a practical and unbiased phenotypic profiling and screening tool for small molecule and RNAi perturbation of specific cellular pathways and time resolution of the TCRP approach can serve as a complement for other existing multidimensional profiling approaches.

The human immune system is highly complex and immune status is associated with disease status, treatment efficiency, and response to external stimuli such as vaccines. For example, the immunophenotyping of leukemia has become indispensable for diagnosis of hematological malignancies. Monitoring T lymphocytes has gained diagnostic importance in the prognosis and prediction of therapies. In this study, a series of T-cell markers previously used in the HIPC (Human Immunology Phenotyping Consortium) project were assessed. Through comprehensive consideration and analysis of the expression of each marker, fluorescence intensity, spectral overlap, and NovoCyte configuration, a 13-color T-cell phenotyping panel was designed. Next, peripheral blood mononuclear cells (PBMCs) were tested on a NovoCyte. Live T cells (CD3+/Aqua−) were identified as either T helper cells (Th) or cytotoxic T cells (Tc) cells by expression of CD4 and CD8 antigens. Next, further T cell subtype was determined by CCR7 and CD45RA: naïve (CD45RA+/CCR7+), effector (TEF, CD45RA+/CCR7−), effector memory (TEM, CD45RA−/CCR7−) and central memory (TCM, CD45RA−/CCR7+). Activation of T cells was assessed by the activation marker HLA-DR. Identification of Th subtypes (Th1, Th2 and Th17) was determined by CXCR3 and CCR6. Regulatory T cells (Treg) were identified as CD4+/CD25+/CD127lo/CCR4+, and memory and naïve Treg cells were further differentiated by CD45RO. With this panel, the phenotype and activation of T cell subtypes can be determined in one run which allows quickly and in-depth analysis of human T lymphocyte subsets. NovoCyte flow cytometer features high resolution of dim signals and flexible configuration and is particularly suitable for multicolor analysis.

No abstract

No abstract

Although apoptosis can be tracked using various biomarker‐based assays, these typically require multiple manual handling steps and only yield endpoint measurements. Requiring just a cell seeding step and a drug addition step, we used the Agilent xCELLigence RTCA eSight to continuously monitor drug‐mediated apoptosis over the course of multiple days. Providing a direct and objective assessment of cell number, cell size, cell‐substrate attachment strength, and cell barrier function, impedance biosensors embedded within the base of eSight microplates quantitatively track early (cell shrinkage) to late (fragmentation) apoptotic events with high analytical sensitivity. Concurrently, eSight captures live cell images in brightfield and three fluorescence channels (red, green, and blue), providing an orthogonal readout of the apoptosis process. By combining the strengths of real‐time impedance monitoring (simplicity, analytical sensitivity, and objectivity) with that of live cell imaging (specificity of the readout), eSight increases the information richness of the apoptosis assay without increasing the workload. Importantly, the drug EC50 values determined using these two approaches are nearly identical, suggesting that eSight can simultaneously provide both the primary and secondary (confirmatory) readouts for apoptosis studies. Corroborating the impedance response with live cell images. Image panels demonstrate the progression of apoptosis 20 and 40 hours after treating A549‐Blue cells with 5.5 μm MG132. White arrows denote large membrane blebs that contain phosphatidylserine in their outer leaflet. Multiplex monitoring of apoptosis in real‐time: combining readouts from cellular impedance and live cell imaging.

No abstract