Calcein AM (Calcein acetoxymethyl ester) is a non-fluorescent compound that passively enters cells. In metabolically active cells, Calcein AM is converted by cytosolic esterases into green fluorescent Calcein. The fluorescent Calcein is retained by live cells with intact membranes. Only cells possessing active cytosolic esterases fluoresce green. This allows for quick and easy detection of metabolically-active (vital) cells in a sample. […]
Natural Killer (NK) Cells are large granular lymphocytes that belong to the innate immune system and make up approximately 10% of circulating lymphocytes. Unlike T cells, NK cells do not express CD3. NK cells are critical for protection from life-threatening infections and are important mediators of antitumor immunity. Rare reports of complete NK-cell deficiencies in humans have resulted in fatal infection during childhood. Uncontrolled or inappropriate NK cell response can lead to pathological conditions such as allograft rejection, graft vs. host disease, diabetes, aplastic anemia, and various autoimmune and neurological diseases. […]
GFP and Determination of Transfection Efficiency Green Fluorescent Protein (GFP) is a 26.9 kDa protein that fluoresces bright green when exposed to blue or ultraviolet light. GFP was first identified as a protein and extracted from the Aequorea victoria jellyfish in 1962 by Osamu Shimomura, et al. The GFP protein was first cloned in 1992 and it was soon confirmed that GFP protein expressed in other organisms generates fluorescence. An area within a cell or tissue is briefly illuminated, causing the GFP protein to fluoresce, allowing GFP-tagged proteins to be identified. In 2008, Osamu Shimomura, Marty Chalfie, and [...]
Autophagy is a normal process by which eukaryotic cells break down out-dated and damaged cellular organelles and proteins to be replaced with new ones. It is also a survival mechanism providing cells with energy and substrates for cellular processes in times of stress and starvation. Autophagy is a multi-step process involving initiation, formation of autophagosomes (vesicles that capture and deliver cytoplasmic material to lysosomes for digestion), maturation, and degradation (Figure 1.) […]
Adipocytes are the primary cells found in adipose (fat) tissue. Adipocytes store energy for the body in the form of triacylglycerol and play a critical role in lipid metabolism and energy regulation. Adipocytes have also been found to express a wide range of cytokines, chemokines, receptors, and cell adhesion molecules capable of inducing inflammation. Though it was once believed that mature adipocytes do not proliferate, a small degree of proliferation has been observed in mature adipocytes in culture. It is likely that overall adipocyte cell number is maintained over time by highly-controlled apoptosis and proliferation. […]
Caspase-3 belongs to a family of evolutionally conserved cysteine proteases that play a key role in regulating programmed cell death, or apoptosis, a normal process required for maintenance of tissue homeostasis and the regulation of physiological functions1. The two main apoptosis activation pathways are the extrinsic and the intrinsic pathways. The extrinsic pathway is activated by the binding of ligands (including TNFα, FasL, and TRAIL) to cell-surface receptors. The intrinsic, or mitochondrial, pathway is typically activated in response to DNA or cellular damage. The convergence of the extrinsic and intrinsic pathways occurs at the proteolytic activation of caspase-34. Once caspase-3 is [...]
Gene expression actualizes the organismal phenotypes encoded within the genome in an environment-dependent manner. Among all encoded phenotypes, cell population growth rate (fitness) is perhaps the most important, since it determines how well-adapted a genotype is in various environments. Traditional biological measurement techniques have revealed the connection between the environment and fitness based on the gene expression mean. […]
Ficoll separation is routinely used to isolate mononuclear cells from bone marrow, peripheral blood, and umbilical cord blood. Monocytes and lymphocytes form a buffy coat under a layer of plasma, where the cells are collected, then washed. Typically, some residual platelets and red blood cells are mixed in with the mononuclear cells. The degree of platelet and red blood cell contamination can vary significantly between donors and operators. The Cellometer Auto 2000 Cell Viability Counter utilizes dual fluorescence counting to generate accurate live cell and dead cell counts with no interference from red blood cells and platelets. Experimental Procedure Combine 20µl [...]
Isolation, Quantitation and Viability Analysis of Neonatal Cardiomyocytes using Cellometer The neonatal rat cardiomyocyte model has been used for many years by researchers studying the heart. In addition to increasing understanding of the morphological, biochemical, and electrophysiological characteristics of the normal heart, neonatal cardiomyocytes have been used to study contraction, ischemia, hypoxia, and the toxicity of different compounds. This model has been used to determine the optimal dosage for certain drugs and to develop and evaluate the efficacy of potential therapeutic agents. Apoptosis, or programmed cell death, has been observed in infarcted and re-perfused myocardium, end stages of heart failure, [...]
Caspase-8 Caspase-8 belongs to a family of evolutionally conserved cysteine proteases that play a key role in regulating programmed cell death or apoptosis. The two main apoptosis activation pathways are the extrinsic and the intrinsic pathways. The extrinsic pathway is activated by the binding of ligands (including TNFα, FasL, and TRAIL) to cell-surface receptors and the conversion of procaspase-8 to the active caspase-8 protease.1 Caspase-8 quickly converts procaspase-3 to active Caspase-3, facilitating many of the cellular and biochemical events of apoptosis. The intrinsic, or mitochondrial, pathway is typically activated in response to DNA or cellular damage and begins with the [...]