Search results for "Tissue Culture Techniques"
showing 10 items of 32 documents
Subcellular fractionation of tissue culture cells.
2003
Cell fractionation techniques include some of the most important and widely used analytical tools in cell and molecular biology, and are essential for the development of cell-free assays that reconstitute complicated cellular processes. In addition to simple gradient systems, this unit discusses the immuno-purification of organelles, in particular endosomes. As antigens, purification can be achieved using endogenous or ectopically expressed proteins, provided that appropriate antibodies are available. Alternatively, tagged proteins can be used, when combined with anti-tag antibodies. Now that sequencing of the genomes of several organisms has been completed, biochemical strategies, and in p…
GABA transporters control GABAergic neurotransmission in the mouse subplate.
2015
The subplate is a transient layer between the cortical plate and intermediate zone in the developing cortex. Thalamo-cortical axons form temporary synapses on subplate neurons (SPns) before invading the cortical plate. Neuronal activity within the subplate is of critical importance for the development of neocortical circuits and architecture. Although both glutamatergic and GABAergic inputs on SPns were reported, short-term plasticity of GABAergic transmission has not been investigated yet. GABAergic postsynaptic currents (GPSCs) were recorded from SPns in coronal neocortical slices prepared from postnatal day 3-4 mice using whole-cell patch-clamp technique. Evoked GPSCs (eGPSCs) elicited b…
Use of cultured human epithelium for coverage: a defect of radial forearm free flap donor site
2010
The radial forearm free flap has been popular in many areas of reconstructive surgery. Despite the many attributes of this flap in maxillofacial reconstruction, one of the disadvantages has been the morbidity of the donor site. Allogeneic cultured epidermis has been successfully applied on large second degree burns and on chronic leg ulcers. Autologous human keratinocytes and fibroblast equivalents can be cultured in-vitro from a small skin sample in order to produce a sufficient amount of epithelial autografts to cover the large defects of third-degree burn wounds. Interestingly, transplanted cultured epidermis retains characteristics of the original donor site. We report a case of a patie…
Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue
2007
Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosi…
Sex Differences in the Neuroadaptations of Reward-related Circuits in Response to Subchronic Variable Stress
2018
Women are twice as likely to be diagnosed with major depressive disorder. However, fewer studies in rodent models of depression have used female animals, leading to a relative lack of understanding of the female brain’s response to stress, especially at a neural circuit level. In this study, we utilized a 6-day subchronic variable stress (SCVS) mouse model and measured novelty suppressed feeding as behavioral criteria to evaluate susceptibility to SCVS in male and female mice. First, we showed that SCVS induced a decrease in latency to eat (susceptible phenotype) in female mice, but not in males (resilient phenotype). After determining behavioral phenotypes, we investigated the firing activ…
Plasticity-Related Gene 1 Affects Mouse Barrel Cortex Function via Strengthening of Glutamatergic Thalamocortical Transmission
2016
Plasticity-related gene-1 (PRG-1) is a brain-specific protein that modulates glutamatergic synaptic transmission. Here we investigated the functional role of PRG-1 in adolescent and adult mouse barrel cortex both in vitro and in vivo. Compared with wild-type (WT) animals, PRG-1-deficient (KO) mice showed specific behavioral deficits in tests assessing sensorimotor integration and whisker-based sensory discrimination as shown in the beam balance/walking test and sandpaper tactile discrimination test, respectively. At P25-31, spontaneous network activity in the barrel cortex in vivo was higher in KO mice compared with WT littermates, but not at P16-19. At P16-19, sensory evoked cortical respo…
N-Valproyl-L-Phenylalanine as new potential antiepileptic drug: Synthesis, characterization and in vitro studies on stability, toxicity and anticonvu…
2013
Valproic acid (VPA) is considered first-line drug in treatment of generalized idiopathic seizures such as absence, generalized tonic-clonic and myoclonic seizures. Among major antiepileptic drugs, VPA is also considered effective in childhood epilepsies and infantile spasms. Due to its broad activity, VPA acts as a mood stabilizer in bipolar disorder and it is useful in migraine prophylaxis. Despite its long-standing usage, severe reactions to VPA, such as liver toxicity and teratogenicity, are reported. To circumvent side effects due to structural characteristics of VPA, we synthesized in good yield a new VPA-aminoacid conjugate, the N-valproyl-L-Phenylalanine, and characterized by FT-IR, …
Decreased Paraoxonase-2 Expression in Human Carotids During the Progression of Atherosclerosis
2008
Objective— Many gene products involved in oxidation and inflammation are implicated in the pathogenesis of atherosclerosis. We investigated paraoxonase 2 (PON2), 5-lipoxygenase (5-LO), and 5-LO activating protein (FLAP) expression and malondialdehyde (MDA) levels in carotid lesions to assess their involvement in plaque formation. Methods and Results— We measured gene expression and MDA levels in atherosclerotic plaques from 59 patients undergoing carotid endarterectomy, and in plaque-adjacent tissue from 41/59 patients. Twenty-three fetal carotids and 6 mammary arteries were also investigated. Real-time polymerase chain reaction and immunohistochemistry revealed decreased PON2 expression i…
Photoreceptor cells display a daily rhythm in the orphan receptor Esrrβ
2015
Purpose Nuclear orphan receptors are critical for the development and long-term survival of photoreceptor cells. In the present study, the expression of the nuclear orphan receptor Esrrβ—a transcriptional regulator of energy metabolism that protects rod photoreceptors from dystrophy—was tested under daily regulation in the retina and photoreceptor cells. Methods The daily transcript and protein amount profiles were recorded in preparations of the whole retina and microdissected photoreceptor cells using quantitative PCR (qPCR) and western blot analysis. Results Esrrβ displayed a daily rhythm with elevated values at night in the whole retina and enriched photoreceptor cells. Daily regulation…
Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: From the in vitro ACE assay to the spontaneously hypertensive rat
2011
Bioactive ACE inhibiting peptides are gaining interest in hypertension treatment. We have designed and screened six synthetic heptapeptides (PACEI48 to PACEI53) based on two hexapeptide leads (PACEI32 and PACEI34) to improve ACE inhibitory properties and assess their antihypertensive effects. ACE activity was assayed in vitro and ex vivo. Selected peptides were administered to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. In vitro cytotoxicity was assessed with the MTT reduction test. The six heptapeptides at low micromolar concentration produced different degrees of in vitro inhibition of ACE activity using the synthetic substrate HHL or the natural subst…