Trisomy 12 assessment by conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and laser scanning cytometry (LSC) in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

Quantitative assessment of pancreatic islets using laser scanning cytometry.

Insulin-dependent (type II) diabetes is characterized by an inability to metabolize glucose, resulting from insufficient insulin function for glucose transport from the blood to tissues. One cause of insufficiency is malfunction of the insulin-producing beta cells within the pancreatic islets. Various compounds to stimulate and restore normal islet function are under development. Zucker diabetic fatty (ZDF) rat animal models are used to measure efficacy of drug candidates, as they show clinical effects similar to those in diabetic patients. Drug effects are evaluated by removing the pancreas from ZDF rats, processing the tissue with paraffin and sectioning it, and then analyzing the sections utilizing antibodies against targeted proteins to quantify morphology and metabolic activity. This protocol describes quantitative analysis of insulin, glucagon, mitochondria (all on a per-islet basis), and insulin-positive proliferating cells in ZDF and lean rat pancreatic tissue sections using the iCyte Imaging Cytometer.

Cytometric assessment of cytostatic and cytotoxic effects of topical glaucoma medications on human epithelial corneal line cells.

BACKGROUND: The long-term-treatment of glaucoma with topical medications is associated with side effects involving cornea damage. We examined the effect of glaucoma topical medications (bimatoprost, travoprost, latanoprost, timolol, betaxolol, dorzolamide, brinzolamide, brimonidine) on growth of cells of three human epithelial corneal lines. METHODS: The cells were cultured in 8-chamber slides, treated with different concentrations of the medications, and fixed at 24, 48, and 72 h. Cell number on slides to estimate viability and growth curves, frequency of apoptosis (FLICA and caspase-3 activation probes), and proliferation (BrdU incorporation assay) were measured by laser scanning cytometry (LSC). RESULTS: Depending on concentration all examined medications induced cell necrosis or apoptosis and suppressed proliferation. Significant variability in proliferation and apoptosis was observed within the same cultures depending on local cell density, with cells in high density areas being more resistant. The data indicate that commonly used topical medications exert cytostatic and cytotoxic effects in cultures of corneal cells and suggest that caution should be exercised in their use, particularly, when the corneal diseases are accompanied by cell proliferation and regeneration, in long-term-treatment. CONCLUSIONS: The present approach of using LSC makes it possible to assess and compare cytostatic and cytotoxic effects of different topical medications on the respective target cells.

Microtransponders, the miniature RFID electronic chips, as platforms for cell growth in cytotoxicity assays.

BACKGROUND: An electronic radio frequency (RF) microchip, the microtransponder (MTP), has been developed as a platform for assays in the fields of genomics and proteomics. Upon activation by light, each MTP provides a unique RF identification (ID) signal that matches a chip to the specific biological material attached to it. The MTP is powered by a photocell and has an antenna that transmits the signal. The aim of the present study was to explore utility of MTPs as a platform for cell growth in cytotoxicity assays. METHODS: The MCF-7, MCF-116, A549, or T-24 cells growing on MTPs placed in petri dishes or slide chambers were cultured untreated or exposed to antitumor drugs topotecan, mitoxantrone, or onconase for up to 4 days. Their attachment to- and growth on- MTPs was assessed by fluorescence microscopy and laser scanning cytometry (LSC) and compared with growth on the dish surface in the MTP neighborhood. The MTPs were fixed in ethanol, stained with propidium iodide (PI), and interrogated in flow in the instrument capable to rapidly (up to 103 MTPs/s) identify their ID signal and measure fluorescence. RESULTS: The cells plated on MTPs exhibited similar attachment properties to those plated in culture dishes. When measured by LSC, they had similar mitotic activity, growth rate, and cell cycle distributions as the cells adhering to the culture dish in the neighborhood of MTPs. The fluorescence intensity of MTPs provided information about the cell number per MTP, which made it possible to assess cell growth rate and monitor the cytostatic/cytotoxic effects of the tested drugs. CONCLUSIONS: The MTP-based system holds promise for the multiplexed cell assays in which numerous different cell lines can be screened for their growth rate or sensitivity while exposed to particular agents in the same vessel. Other advantages of the system are the rapidity of the screening and a very large number of ID codes. Because many cell lines/types can be assayed in a single dish, the system also offers cost savings on tissue culture reagents.

Method for assessment of viability and morphological changes of bacteria in the early stage of colony formation on a simulated natural environment.

A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.

Comparative analysis of the effects of sample source and test methodology on the assessment of protein expression in acute myelogenous leukemia.

Numerous studies have analyzed the expression and prognostic importance of various proteins in acute myelogenous leukemia (AML). We sought to determine whether the sample source and methodology used to measure protein expression affect the results obtained. To determine the importance of sample source, we used Western blotting to compare the expression of eight proteins and phosphoproteins in the leukemia blast-enriched fraction of 118 blood- and 108 marrow-derived samples, including 37 paired samples. To determine the importance of methodology, the expression of five proteins was measured in 20 paired samples by Western blotting, laser scanning cytometry (LSC), and flow cytometry. The mean expression and range of expression in blood- and marrow-derived samples were statistically identical for all eight proteins. Expression measurements for the 37 paired blood and marrow samples also had very high statistical correlation. The LSC and flow cytometry data had the highest concordance when compared using Kolmogorov-Smirnoff D-stats (range of R values, 0.8-1.0). High concordance was also observed between the LSC and flow cytometry results when the percentage of cells positive for expression was dichotomized into positive or negative expression. However, there was less correlation between LSC and flow cytometry when the actual percentages of positive cells were compared. The majority of discordant situations involved samples that were positive by flow cytometry but negative by LSC. The correlation between Western blotting signal intensity and the percentage of expression-positive cells measured by LSC or flow cytometry varied by protein but was limited when there was little heterogeneity in expression by either method. In conclusion, provided that leukemia blast-enriched fractions were analyzed, the blood- and marrow-derived samples had identical protein expression. There was good concordance of results between flow cytometry and LSC, which share similar technology, but more limited correlation between these methods and Western blotting.

Assessment of a new technique combining a viability test, whole-cell hybridization and laser-scanning cytometry for the direct counting of viable Enterobacteriaceae cells in drinking water.

A new direct approach, called direct viable count (DVC)-FISH-ScanRDI, combining viability measurement, specific detection and sensitive enumeration of highly diluted Enterobacteriaceae cells, was assessed during the summer in water samples from a North American drinking water treatment plant and its distribution system. Major results of this field investigation show a higher sensitivity of the DVC-FISH-ScanRDI approach in enumerating viable Enterobacteriaceae cells in distributed drinking water, relative to a culture-based method, and the increased concentration of viable but non-culturable (VBNC) Enterobacteriaceae cells in distributed water for temperatures above 18 degrees C.