A Novel Recombinant Vitronectin Variant Supports the Expansion and Differentiation of Pluripotent Stem Cells in Defined Animal-Free Workflows.

An essential aspect of harnessing the potential of pluripotent stem cells (PSCs) and their derivatives for regenerative medicine is the development of animal-free and chemically defined conditions for ex vivo cultivation. PSCs, including embryonic and induced PSCs (iPSCs), are in the early stages of clinical trials for various indications, including degenerative diseases and traumatic injury. A key step in the workflows generating these cells for more widespread clinical use is their safe and robust ex vivo cultivation.

Revolutionizing Cancer Treatments through Stem Cell-Derived CAR T Cells for Immunotherapy: Opening New Horizons for the Future of Oncology.

Recent advances in cellular therapies have paved the way for innovative treatments of various cancers and autoimmune disorders. Induced pluripotent stem cells (iPSCs) represent a remarkable breakthrough, offering the potential to generate patient-specific cell types for personalized as well as allogeneic therapies. This review explores the application of iPSC-derived chimeric antigen receptor (CAR) T cells, a cutting-edge approach in allogeneic cancer immunotherapies.

Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs.

ELISpot and flow cytometry are two methods often utilized side-by-side for detecting secreted and intracellular cytokines, respectively. Each application has its own advantages and challenges. ELISpot is more sensitive compared to ELISA and appears to be more consistent in detecting IL-10 production than flow cytometry.

The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the β-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity.

In Situ Hybridization (ISH) Combined with Immunohistochemistry (IHC) for Co-detection of EGFR RNA and Phosphorylated EGFR Protein in Lung Cancer Tissue.

Detection of phosphorylated proteins in tissue sections using immunohistochemistry (IHC) is a challenging task. The absence of tissue staining may be caused by either a lack of protein expression or a lack of protein activation via its phosphorylation. To address this problem, we employed Integrated Co-detection Workflow (ICW) protocol to analyze lung cancer tissue sections by combining in situ hybridization (ISH) with IHC.

In Situ Hybridization (ISH) Combined with Immunocytochemistry (ICC) Co-detection of Phosphorylated EGFR in A431 Cultured Cells.

Antibodies have been commonly used to study protein phosphorylation since the first phospho-specific antibody was described in 1981. Antibodies can be developed so that they specifically recognize phosphorylated areas of particular proteins. In situ hybridization (ISH) is the technique where specific RNA or DNA molecules can be detected in a single cell without the need for antibodies.

Labeling of Phospho-Specific Antibodies with oYo-Link® Epitope Tags for Multiplex Immunostaining.

Spatial proteomics has recently garnered significant interest, as it offers to provide unprecedented insight into biological processes in both health and disease, by connecting protein expression patterns from the subcellular level to the tissue or even organism level. These high-content approaches generally rely on a high degree of multiplexing, whereby multiple proteins can be detected simultaneously. The most versatile multiplexing approaches utilize antibodies to confer specificity for various intracellular proteins of interest.

[Possibilities of complex rehabilitation of young children with epilepsy and movement disorders].

Unlabelled: Epilepsy is a chronic disease characterized by recurrent, mostly unprovoked seizures with impaired motor, autonomic, mental or mental functions that occur as a result of excessive neuronal discharges in the gray matter of the cerebral cortex. The problem of the activity of medical rehabilitation for epilepsy in the professional community remains debatable, despite the obviousness of the arguments and judgments presented.

Purpose Of The Study: Development of an effective and safe complex for the rehabilitation of young children with epilepsy, accompanied by impaired movement function.

Inhibition of DAGLβ as a therapeutic target for pain in sickle cell disease.

Sickle cell disease (SCD) is the most common inherited disease. Pain is a key morbidity of SCD and opioids are the main treatment but their side effects emphasize the need for new analgesic approaches. Humanized transgenic mouse models have been instructive in understanding the pathobiology of SCD and mechanisms of pain.

A Never-Ending Journey in Search for Novel Cell Biology Techniques.

Cell techniques undergo rapid advancement across different areas of biomedical research [...

The bivalent ligand, MMG22, reduces neuropathic pain after nerve injury without the side effects of traditional opioids.

Functional interactions between the mu opioid receptor (MOR) and the metabotropic glutamate receptor 5 (mGluR5) in pain and analgesia have been well established. MMG22 is a bivalent ligand containing MOR agonist (oxymorphamine) and mGluR5 antagonist (MPEP) pharmacophores tethered by a 22-atom linker. MMG22 has been shown to produce potent analgesia in several models of chronic inflammatory and neuropathic pain (NP).

Essential Controls for ELISpot Assay.

Nonspecific staining in ELISpot assay is a major obstacle in accurate quantification of experimental data. The appearance of nonspecific spots may be caused by different factors including cell- and immunoassay-related issues. In our study, we have shown that nonspecific spots can result from either cells or their debris sticking to the membranes in ELISpot plates, as well as by impurities in wash buffers and precipitation of aggregated detection antibodies.

Atlas of Immune Cell Populations of the Inflamed Mammalian CNS.

Two processes are known to take place during neuroinflammation: (i) resident immune cells are activated and (ii) inflammatory leukocytes in the periphery begin to infiltrate the central nervous system (CNS).[…].

What Makes Cells Different from Other Open Access Journals.

In 2011, I was invited to serve as the Editor-In-Chief for Cells, which back then was a "new kid on the block" among open access (OA) journals.[..

Express γ-H2AX Immunocytochemical Detection of DNA Damage.

DNA can be damaged by many environmental factors including chemical agents and ionizing radiation which induce the formation of DNA double-stranded breaks (DSBs). If DSBs are not repaired in a timely fashion this may cause the disruption of genome integrity, which can result in cancer development. Typically, DSBs are followed by phosphorylation of histone protein H2AX, a member of the H2A family.

Automated High-Content Screening of γ-H2AX Expression in HeLa Cells.

Due to their inherent nature, DNA strands can be easily broken by various environmental factors including chemical agents and ionizing radiation. Unrepaired DNA double-stranded breaks (DSBs) may result in genetic instability and have a strong negative impact on the integrity of the genome. It has been found that DSBs are always followed by phosphorylation of histone protein H2AX, a member of the H2A family, and immunocytochemical detection of phosphorylated H2AX (referred to as γ-H2AX) is one of the frequently used techniques for assessing DNA damage.

Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry.

Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific.

High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP.

A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody.

Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX Using Primary Antibodies Raised in the Same Host Species.

Multiplex staining of cell and tissue sections with antibodies raised in the same host species is a serious challenge because of unwanted but inevitable cross-reactivity of secondary antibodies with irrelevant primary antibodies. Several techniques can be used to overcome this obstacle including direct labeling of primary antibodies with fluorescent tags and using tyramide signal amplification. Unfortunately these techniques either lack sensitivity, or require a long multistep protocol which can cause physical damage of specimens.

Membrane Microplates for One- and Two-Color ELISPOT and FLUOROSPOT Assays.

Membranes are widely used as protein blotting matrices for a large variety of research applications including western blotting and enzyme-linked immunospot assay (ELISPOT). The largest advantage of using membranes versus solid plastic support is the porosity of membranes allowing for immobilization of high concentrations of proteins and antibodies which, in turn, increases the sensitivity of detection. Similar to plastic surfaces, polyvinylidene difluoride (PVDF) and nitrocellulose membranes create good microenvironment for live cells cultured in vitro and do not interfere with cellular physiology.

Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays.

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays.

Putative kappa opioid heteromers as targets for developing analgesics free of adverse effects.

It is now generally recognized that upon activation by an agonist, β-arrestin associates with G protein-coupled receptors and acts as a scaffold in creating a diverse signaling network that could lead to adverse effects. As an approach to reducing side effects associated with κ opioid agonists, a series of β-naltrexamides 3-10 was synthesized in an effort to selectively target putative κ opioid heteromers without recruiting β-arrestin upon activation. The most potent derivative 3 (INTA) strongly activated KOR-DOR and KOR-MOR heteromers in HEK293 cells.

Bivalent ligands that target μ opioid (MOP) and cannabinoid1 (CB1) receptors are potent analgesics devoid of tolerance.

Given that μ opioid (MOP) and canabinoid (CB1) receptors are colocalized in various regions of the central nervous system and have been reported to associate as heteromer (MOP-CB1) in cultured cells, the possibility of functional, endogenous MOP-CB1 in nociception and other pharmacologic effects has been raised. As a first step in investigating this possibility, we have synthesized a series of bivalent ligands 1-5 that contain both μ agonist and CB1 antagonist pharmacophores for use as tools to study the functional interaction between MOP and CB1 receptors in vivo. Immunofluorescent studies on HEK293 cells coexpressing both receptors suggested 5 (20-atom spacer) to be the only member of the series that bridges the protomers of the heteromer.

An immunocytochemical-derived correlate for evaluating the bridging of heteromeric mu-delta opioid protomers by bivalent ligands.

Bivalent ligands that contain two pharmacophores linked by a spacer are promising tools to investigate the pharmacology of opioid receptor heteromers. Evidence for occupation of neighboring protomers by two phamacophores of a single bivalent ligand (bridging) has relied mainly on pharmacological data. In the present study, we have employed an immunocytochemical correlate to support in vivo biological studies that are consistent with bridging.

Combining ELISPOT and ELISA to measure amounts of cytokines secreted by a single cell.

Enzyme-linked immunospot (ELISPOT) assay allows for the determination of the frequency of -cytokine-secreting cells, but does not answer the question of how much cytokine is secreted per cell. In our study, we combined ELISPOT and ELISA assays and developed a protocol to calculate the amount of IFN gamma secreted by each cell. A suspension of human peripheral blood mononuclear cells was split into two pools and cells from one pool were cultured in a regular ELISPOT plate, whereas cells from the other pool were cultured in an uncoated, "blank," ELISPOT plate.