flow-cytometry-protocols-for-surface-intracellular-antigen-analyses

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

University of Freiburg

In an unselected consecutive postmortem series, 512% of the adrenals showed cortical nodules with diameters up to 1 mm in 13%, between 1 and 2 mm in 28% and < 2 mm in 12%. In 22% of cases only one nodule was present; whereas in 15% two nodules and in 17% more than three nodules were found. A cortical adenoma was detected in 5% of cases. Adenomas were smaller than 6 mm in 1% and < 15 mm also in 1% of the entire collection. For differentiation of nodules criteria listed in Table 4 were used. Correlation with clinical data revealed a significantly higher number of nodules and an adenoma more frequently in hypertensive patients. Metastases were found in 19% of all cases with malignant tumors. Twenty-three percent of the metastases were smaller than 1 mm and 20% were < 10 mm in diameter. Adrenal metastases usually indicated generalized dissemination of the tumor.

Hyperplastic and Tumorous Lesions of the Adrenals in an Unselected Autopsy Series.

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.

Genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility in primary ciliary dyskinesia (PCD). The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Here, we demonstrate that large genomic deletions, as well as point mutations involving LRRC50, are responsible for a distinct PCD variant that is characterized by a combined defect involving assembly of the ODAs and IDAs. Functional analyses showed that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5- and DNAI2-containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia. On the basis of these findings, we assume that LRRC50 plays a role in assembly of distinct dynein-arm complexes.

Deletions and point mutations of LRRC50 cause primary ciliary dyskinesia due to dynein arm defects.

Aminoacylase 1 (ACY1) deficiency is a recently described inborn error of metabolism. Most of the patients reported so far have presented with rather heterogeneous neurologic symptoms. At this moment, it is not clear whether ACY1 deficiency represents a true metabolic disease with a causal relationship between the enzyme defect and the clinical phenotype or merely a biochemical abnormality. Here we present a patient identified in the course of selective screening for inborn errors of metabolism (IEM). The patient was diagnosed with autistic syndrome and admitted to the Children's Memorial Health Institute (CMHI) for metabolic evaluation. Organic acid analysis using gas chromatography-mass spectrometry (GC-MS) revealed increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, glycine, leucine, isoleucine, and valine. In Epstein-Barr virus (EBV)-transformed lymphoblasts, ACY1 activity was deficient. The mutation analysis showed a homozygous c.1057C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation and had normal results in the organic acid analysis using GC-MS. This article reports the findings of an ACY1-deficient patient presenting with autistic features.

Aminoacylase 1 deficiency associated with autistic behavior.

A complete calibration method to characterize a static planar two-dimensional detector for use in X-ray diffraction at an arbitrary wavelength is described. This method is based upon geometry describing the point of intersection between a cone's axis and its elliptical conic section. This point of intersection is neither the ellipse centre nor one of the ellipse focal points, but some other point which lies in between. The presented solution is closed form, algebraic and non-iterative in its application, and gives values for the X-ray beam energy, the sample-to-detector distance, the location of the beam centre on the detector surface and the detector tilt relative to the incident beam. Previous techniques have tended to require prior knowledge of either the X-ray beam energy or the sample-to-detector distance, whilst other techniques have been iterative. The new calibration procedure is performed by collecting diffraction data, in the form of diffraction rings from a powder standard, at known displacements of the detector along the beam path.

Complete elliptical ring geometry provides energy and instrument calibration for synchrotron-based two-dimensional X-ray diffraction.

The development of a technique for following in situ the reactions of solids with alkali metal/ammonia solutions, using time-resolved X-ray diffraction methods, reveals high-temperature superconducting ammonia-rich intercalates of iron selenide which reversibly absorb and desorb ammonia around ambient temperatures.

Ammonia-rich high-temperature superconducting intercalates of iron selenide revealed through time-resolved in situ X-ray and neutron diffraction.

A new technique combining in situ X-ray diffraction using synchrotron radiation and infrared thermal imaging is reported. The technique enables the application, generation and measurement of significant thermal gradients, and furthermore allows the direct spatial correlation of thermal and crystallographic measurements. The design and implementation of a novel furnace enabling the simultaneous thermal and X-ray measurements is described. The technique is expected to have wide applicability in material science and engineering; here it has been applied to the study of solid oxide fuel cells at high temperature.

A novel high-temperature furnace for combined in situ synchrotron X-ray diffraction and infrared thermal imaging to investigate the effects of thermal gradients upon the structure of ceramic materials.

High-throughput processing of parallel-beam X-ray tomography at synchrotron facilities is lacking a reliable and robust method to determine the center of rotation in an automated fashion, i.e. without the need for a human scorer. Well-known techniques based on center of mass calculation, image registration, or reconstruction evaluation work well under favourable conditions but they fail in cases where samples are larger than field of view, when the projections show low signal-to-noise, or when optical defects dominate the contrast. Here we propose an alternative technique which is based on the Fourier analysis of the sinogram. Our technique shows excellent performance particularly on challenging data.

Christina Reinhard

Emmy Noether-Group for Stem Cell Biology,
Department of Molecular Embryology,
Institute of Anatomy and Cell Biology,
Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology

University of Freiburg

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types