John T. Ransom

HTPS Flow Cytometry: A Novel Platform for Automated High Throughput Drug Discovery and Characterization

The flow cytometer is unique among biomedical analysis instruments because it makes simultaneous and multiple optical measurements on individual cells or particles at high rates. High throughput flow cytometry represents a potentially important multifactorial approach for screening large combinatorial libraries of compounds. Limiting this approach has been the availability of instrumentation and methods in flow cytometry for automated sample handling on the scale required for drug discovery applications. Here, we describe an automated system in which a novel patented fluidics-based pharmacology platform, the HTPS (High Throughput Pharmacological System), is coupled to a flow cytometer using a recently described plug flow-coupling valve technology. Individual samples are aspirated sequentially from microplate wells and delivered to a flow cytometer for rapid multiparametric analysis. For primary screening to detect and quantify cell fluorescence in endpoint assays, a high-speed no-wash protocol enabled processing of 9-10 cell samples/min from 96-well microplates. In an alternate primary screening format, soluble receptor ligands were sampled from microplate wells at rates of 3-4 samples/minute and successfully assessed for the ability to elicit intracellular calcium responses. Experiments with fluorescent beads validated the accurate automated production by the HTPS of exponential and linear gradients of soluble compounds. This feature enabled rapid (2- to 3-min) characterization of the intracellular calcium dose response of myeloid cells to formyl peptide as well as the quantitative relationship between formyl peptide receptor occupancy and cell response. HTPS flow cytometry thus represents a powerful high throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized.

Flow cytometric ratio analysis of the Hoechst 33342 emission spectrum: multiparametric characterization of apoptotic lymphocytes

The apoptotic response to various stimuli is an important part of immune regulation, and the ability to identify apoptotic lymphocytes within a complex population is a prerequisite to a more detailed understanding of its role in vivo, We described a flow cytometric technique which utilizes viable cells and enables simultaneous identification of apoptotic cells and analyses of immunophenotype, cell cycle progression, membrane integrity and light scatter properties. It is based upon analysis of two regions of the emission spectrum of the DNA-binding vital dye hoechst 33342. We established a precise correlation between the ratio of red to blue fluorescence emission and apoptosis based upon nuclear morphology and the presence of characteristic DNA degradation patterns. In human peripheral blood lymphocytes (PBL) and mouse thymocytes we incorporated light scatter properties, cell cycle stage, relevant cell surface immunophenotypic markers (CD25 or CD4) and CD8) and a marker of plasma membrane integrity (merocyanine 540) to enable multiparametric phenotyping of apoptotic cells. We show that staurosporine-induced apoptosis of ConA-stimulated PBL is not correlated with cell cycle stage but is selective for activated cells since the frequency of large, CD25+ cells is decreased by staurosporine. Dexamethasone and ionomycin differ in their ability to induce apoptosis selectively in murine thymocyte subsets. Dexamethasone kills a broad spectrum of the CD4/8 immunophenotypes with no selectively for cell cycle stage. Ionomycin selectively deplete CD4+8+ cells, especially those in the Go/G1 region of the cell cycle, and spared CD4-8+ cells. This technique is broadly advantageous for in vitro and ex vivo models of apoptosis in that it interrogates individual viable cells and correlates membrane and nuclear apoptotic changes with standard flow cytometric immunophenotyping.

High-Throughput Microfluidic Mixing and Multiparametric Cell Sorting for Bioactive Compound Screening

HyperCyt®, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt® configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with ~ 10,000 cells analyzed per reaction. Cell Ca 2+ responses were detected to as little as 10-11 Mpeptide with no detectable carryover between samples at up to 10-7 M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day. (Journal of Biomolecular Screening 2004:103-111)

Flow cytometry systems for drug discovery and development

HT-PS is a fluidics-based pharmacology platform that uses viable cells and test compounds to rapidly identify active compounds and immediately determine their potency and specificity. Axiom employs this proprietary flow-through fluidics system coupled to a flow cytometer (FCM) as a detection system. Integration of FCM was enabled through a Plug-Flow Coupler (PFC) device that allows mixtures of cells and test compounds to be delivered to the FCM as discrete plugs of samples under positive air pressure. An FCM detector provides the advantages of multi parametric measurements and multiplexed, single cell analyses. Assays that combine two or more compatible, fluorescent bioresponse indicators simultaneously, such as measurements of intracellular pH and Ca2+, are possible. Alternatively, measurements of one or more bioresponses can be performed on several distinct cell populations individually stained with uniquely addressable fluorescent chromophores. These formats enable multiple experiments on a single sample and provide high content information thereby greatly increasing decision-making power regarding the activity, potency and selectivity of a test compound. Development of significant data with several hundred cells enables reduction in all requisite sample volumes. The PFC enables FCM sample analysis rates of at least 10 samples/minute. The data will illustrate HT-PS/PFC/FCM utility in the drug discovery arena.

Isolation of subclones with enhanced Ca2+ response homogeneity by flow cytometric selection of single cells during a ligand-activated Ca2+ response

In addition to heterogeneity within a cell population in terms of morphologic phenotypes, there can be heterogeneity in terms of response phenotypes. Such response heterogeneity can result in misleading or false-negative experimental results because the signal produced by a minor subset of cells in a population can be difficult to detect with assays that analyze the average response of the whole population. This report describes a technique that enables the investigator (i) to identify whether a subset of a population mobilizes Ca 2+ in response to a stimulus and (ii) to select and subclone cells from the responsive subset. The subcloning procedure can be performed in a manner that either controls for or disregards the kinetics of the response. The resultant subclones exhibit improved response homogeneity detected at the single cell level which is also manifested as an increased signal-to-noise ratio when the average response of the entire subclone population is studied. This technique presents an opportunity to manipulate the existing response heterogeneity toward the experimental requirements of the investigator. Results obtained with two neuronal cell lines are illustrated and other potential applications of the response-dependent subcloning procedure are discussed.