Ger van den Engh

Flow cytometry and cell sorting

Flow cytometry and cell sorting are well-established technologies in clinical diagnostics and biomedical research. Heterogeneous mixtures of cells are placed in suspension and passed single file across one or more laser interrogation points. Light signals emitted from the particles are collected and correlated to entities such as cell morphology, surface and intracellular protein expression, gene expression, and cellular physiology. Based on user-defined parameters, individual cells can then be diverted from the fluid stream and collected into viable, homogeneous fractions at exceptionally high speeds and a purity that approaches 100%. As such, the cell sorter becomes the launching point for numerous downstream studies. Flow cytometry is a cornerstone in clinical diagnostics, and cheaper, more versatile machines are finding their way into widespread and varied uses. In addition, advances in computing and optics have led to a new generation of flow cytometers capable of processing cells at orders of magnitudes faster than their predecessors, and with staggering degrees of complexity, making the cytometer a powerful discovery tool in biotechnology. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors that contribute to the performance of these instruments. The remaining sections will then be divided into clinical- and research-based applications of flow cytometry and cell sorting, highlighting salient studies that illustrate the versatility of this indispensable technology.

Distribution of olfactory receptor genes in the human genome

We demonstrate that members of the olfactory receptor (OR) gene family are distributed on all but a few human chromosomes. Through FISH analysis, we show that OR sequences reside at more than 25 locations in the human genome. Their distribution is biased for terminal bands. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence-relationships are indicative of the inter- and intrachromosomal duplications responsible for OR family expansion. The human genome has accumulated a striking number of dysfunctional copies: 72% of the sequences are pseudogenes. ORF-containing sequences predominate on chromosomes 7,16 and 17.

TRAPPING OF DNA IN NONUNIFORM OSCILLATING ELECTRIC FIELDS

DNA molecules can be manipulated in aqueous solution in a manner analogous to optical trapping. Due to the induction of an electric dipole, DNA molecules are pulled by a gradient force to regions of high electric field strength. Molecules can be locally trapped in an oscillating field using strips of very thin gold film to generate strong electric fields with steep gradients. Spatial control over the trapped molecules is achieved because they are confined to a width of approximately 5 microm along the edges of the gold-film strips. By mixing static and oscillating electric fields, trapped molecules can be moved from one edge to another or made to follow precise trajectories along the edges. This phenomenon should be useful in microdevices for manipulation of small quantities or single molecules of DNA.

Trapping of DNA by dielectrophoresis.

Under suitable conditions, a DNA molecule in solution will develop a strong electric dipole moment. This induced dipole allows the molecule to be manipulated with field gradients, in a phenomenon known as dielectrophoresis (DEP). Pure dielectrophoretic motion of DNA requires alternate current (AC) electric fields to suppress the electrophoretic effect of the molecules net charge. In this paper, we present two methods for measuring the efficiency of DEP for trapping DNA molecules as well as a set of quantitative measurements of the effects of strand length, buffer composition, and frequency of the applied electric field. A simple configuration of electrodes in combination with a microfluidic flow chamber is shown to increase the concentration of DNA in solution by at least 60‐fold. These results should prove useful in designing practical microfluidic devices employing this phenomenon either for separation or concentration of DNA.

High-speed cell sorting: fundamentals and recent advances

Cell sorters have undergone dramatic technological improvements in recent years. Driven by the increased ability to differentiate between cell types, modern advances have yielded a new generation of cytometers, known as high-speed cell sorters. These instruments are capable of higher throughput than traditional sorters and can distinguish subtler differences between particles by measuring and processing more optical parameters in parallel. These advances have expanded their use to facilitate genomic and proteomic discovery, and as vehicles for many emerging cell-based therapies. High-speed cell sorting is becoming established as an essential research tool across a broad range of scientific fields and is poised to play a pivotal role in the latest therapeutic modalities.

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry

Submicron‐sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high‐throughput, multi‐parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high‐resolution flow cytometry‐based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high‐resolution flow cytometry‐based particle quantification and characterization. Increasing concentrations of submicron‐sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high‐resolution flow cytometric sorting of submicron‐sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high‐resolution flow cytometric sorting of different EV subsets is feasible.

Quantification by flow cytometry of chromosome-17 deletions in Smith-Magenis syndrome patients

Abstract We have used bivariate flow karyotyping to quantify the deletions involving chromosome 17 in sixteen patients with Smith-Magenis syndrome (SMS). The fluorescence intensities of mitotic chromosomes stained with Hoechst 33258 and chromomycin were quantified in a dual-beam flow cytometer. For each patient, the position of the peak representing the deleted chromosome 17 was compared to those of the normal homologs of an unaffected parent. The patients could be classified into four groups based on the size of their deletions. The deletions ranged from ∼9–10 Mb (∼10–11% of the chromosome) to below the detection limit of the technique (2 Mb). Different deletion sizes were detected among patients whose high-resolution banding results were similar. Some deletions detected by banding were not detected by flow analyses. Deletion estimates are largely consistent with the results of molecular analyses. Patients with larger deletions that extend into band 17p12 have abnormal electrophysiologic studies of peripheral nerves. Deletion size does not appear to correlate with the degree of mental retardation, presence of behavioral abnormalities, craniofacial anomalies or common skeletal findings in SMS. By identifying patients with varying deletion sizes, these data will aid the construction of a long-range deletion-based map of 17p11.2 and identification of the genes involved in this syndrome.

Fluorescence spectra of DNA dyes measured in a flow cytometer

The spectral properties of fluorescent dyes often vary with experimental conditions. Spectral changes contain information about the particles to which the dye is bound and can have practical consequences for the design of flow cytometry experiments. It is therefore desirable to have spectral information obtained under conditions that approach those under which the measurements in a flow cytometer are performed. This report describes the measurement of fluorescence spectra in a flow cytometer. We have attached a monochromator to one of the detector ports of our flow cytometer. The monochromator scans continuously over a range of wavelengths. For each measured particle, the monochromator wavelength is recorded along with the fluorescence intensity at that wavelength. Other fluorescence and scatter signals can also be recorded to distinguish different particle populations within the sample. This method for measurement of fluorescence spectra has advantages over conventional fluorimetry in that it distinguishes between bound and unbound dye and that spectral properties of different particle populations within a single sample can be separately reconstructed. Fluorescence spectra of common DNA dyes bound to chromatin are presented. A shift to the red in Hoechst 33258 fluorescence with increasing dye concentration is demonstrated. The spectra of DNA-bound Hoechst and ethidium bromide are compared with those of the free dyes in solution. Energy transfer between several pairs of DNA dyes is demonstrated using spectral measurements. We also find slight spectral variations between the different human chromosomes dualstained with Hoechst and chromomycin.

Analysis and sorting of prostate cancer cell types by flow cytometry

Prostate tumor heterogeneity as manifested by differential expression of markers can be attributed to multiple types of cancer cells populating a tumor. Does the composition differ between primary tumor and metastasis? How can one isolate the different cancer cell types to study? What is the relationship among cancer cell types?

Changes in cell surface molecules associated with in vitro culture of prostatic stromal cells

Prostate stromal cells can be readily cultured in vitro. Are these proliferating cells representative of stromal cells in situ? Since the expression of cell surface molecules, like the cluster of differentiation (CD) antigens, can be affected by changes in physiological conditions cultured stromal cells may differ from uncultured stromal cells in their complement of CD molecules.