Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells.

Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM).

Why to compare absolute numbers of mitochondria.

Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry.

NF-κB essential modulator (NEMO) interaction with linear and lys-63 ubiquitin chains contributes to NF-κB activation.

The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation.

Dephosphorylation of Carma1 by PP2A negatively regulates T-cell activation.

The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane.

Differences in the kinetics of gamma-H2AX fluorescence decay after exposure to low and high LET radiation.

Purpose: In order to obtain more insight into heavy ion tumour therapy, some features of the underlying molecular mechanisms controlling the cellular response to high linear energy transfer (LET) radiation are currently analysed.

Materials And Methods: We analysed the decay of the integrated fluorescence intensity of gamma-H2AX (phosphorylated histone H2AX) which is thought to reflect the repair kinetics of radiation-induced DNA double-strand breaks (DSB) using Laser-Scanning-Cytometry. Asynchronous human HeLa cells were irradiated with a single dose of either 1.

Diversity of bacteria growing in natural mineral water after bottling.

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes.

The rate of extrachromosomal homologous recombination within a novel reporter plasmid is elevated in cells lacking functional ATM protein.

Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially.

Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells.

The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C (RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant.

Quantification of temporary and permanent subpopulations of bull sperm by an optimized SYBR-14/propidium iodide assay.

Background: The quality of bull sperm is a key factor in the field of controlled reproduction. Viability-testing is an important aspect of sperm quality definition, especially after cryopreservation where multiple factors such as handling, freeze-thaw cycle, and preservation media, have an impact on the metabolic and functional state of sperm cells.

Methods: We investigated the commonly used SYBR-14/propidium iodide (PI) assay to obtain functional information about sperm-dye and dye-dye interactions.

Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region.

The secreted glycoprotein WNT1 is expressed in the caudal midbrain and is essential for proper development of the entire mid-/hindbrain region. To get better insights into Wnt1 function in the mid-/hindbrain region, we ectopically expressed Wnt1 under the control of the endogenous En1 promoter, thereby extending Wnt1 expression rostrally into the anterior midbrain and caudally into rhombomere 1. In these transgenic mice, the position of the mid-/hindbrain organizer is not altered and pattern formation is not changed.

Detection methods for the expression of the dissimilatory copper-containing nitrite reductase gene (DnirK) in environmental samples.

In situ assays, based on monoclonal antibodies (mAbs), were developed to study the microbial expression of the bacterial dissimilatory copper-containing nitrite reductase gene (DnirK), one of the key enzymes involved in denitrification, in different ecosystems. With a combination of an anti-DnirK mAb and phylogenetic oligonucleotide probes, it is possible to bring structural and functional aspects of microbial communities together. To perform a double labelling, yielding a high signal strength for both the oligonucleotide and the antibody, cells have to be labelled with the oligonucleotide first followed by immunostaining.