Imaging and Flow Cytometry: Competing or Complementary Technologies?

Introduction This presentation will follow the pathways to convergence of two very complementary technologies - flow cytometry and imaging. Interestingly, each technology has evolved and matured almost exclusively isolated from the other. in the past several years it has become increasingly clear that these technologies are able to deliver complementary solutions and that when combined the end value of each exceeds the sum of the individual components.History of development Flow cytometry has a rich history over the past 40 years. Ironically, as a technology, flow cytometry was almost a consolation prize to its imaging cousins. in fact, it seems that it might only have been because imaging solutions proved to be technologically difficult that flow cytometry not only thrived, but left the imaging field well behind for nearly 20 years. Initial interest in evaluating differences between normal and cancer cells was almost exclusively attempted by imaging techniques.</jats:p

Applications of <em>in situ </em>proximity ligation assays for cancer research and diagnostics [Elektronisk resurs]

In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.     Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.    A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry.  </p

Smooth muscle dysfunction occurs independently of impaired endothelium-dependent dilation in adults at risk of atherosclerosis

AbstractObjectives. We sought to assess smooth muscle function in adults at risk for atherosclerosis.Background. Previous studies in subjects at risk for atherosclerosis have demonstrated arterial endothelial dysfunction, with reduced vasodilator responses after pharmacologic or physiologic stimulation of endothelial nitric oxide (NO). Most have also shown a slight but nonsignificant impairment of vasodilation in response to exogenous sources of NO, such as nitroglycerin (NTG). We hypothesized that NTG responses might be reduced in a large number of consecutively studied adults at risk for atherosclerosis, independent of any impaired endothelium-dependent responses, consistent with concomitant smooth muscle dysfunction.Methods. Using high resolution ultrasound, the dilator response of the brachial artery to 400 μg of sublingual NTG was measured in 800 asymptomatic subjects. Subjects were also assessed for a history of vascular risk factors, blood pressure, total serum cholesterol and flow-mediated endothelium-dependent dilation (EDD).Results. We studied 317 men and 483 women, 38 ± 17 years old (mean ± SD, range 15 to 76). The mean cholesterol level was 5.2 ± 1.3 mmol/liter, and there were 126 smokers and ex-smokers (16 ± 9 mean pack-years) and 105 diabetic subjects. On univariate analysis, a reduced vasodilator response to NTG was associated with high cholesterol, cigarette smoking, diabetes mellitus, increasing age, male gender, larger vessel size and reduced EDD (p ≤ 0.01 for all). On multivariate analysis, diabetes, larger vessel size and reduced EDD were all independently associated with impaired NTG-related vasodilation (p ≤ 0.001 for all). In the 574 nondiabetic subjects who had never smoked cigarettes, the independent relation between EDD and NTG responses was still observed (r = 0.24, p = 0.01).Conclusions. The vasodilator response to exogenous NO is impaired in asymptomatic subjects with reduced EDD, consistent with smooth muscle dysfunction in adults at risk for atherosclerosis