absence Protocols

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+ sensitive acidic form. - [Read Assay of Intracellular Free Calcium in Suspended B Cells]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Protocol for a method which assesses concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. Signalling Gateway - [Read Assay of Intracellular Free Calcium in Suspended B Cells PDF]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+- sensitive acidic form. - [Read Assay of Intracellular Free Calcium in Suspended B Cells Protocol]

Category: Immunology: B Cell Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+- sensitive acidic form. - [Read Assay of Intracellular Free Calcium in Suspended B Cells Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Human A431 cells and mouse 3T3 cells are exposed in culture to UV light both in the presence and absence of test compound.  Phototoxicity is expressed as a decrease in cell viability as determined by the MTT assay. - [Read Cell Culture Phototoxicity Test Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

In vitro differentiation of ES cells occurs when the cells are allowed to aggregate in suspension culture in the absence of mouse embryonic fibroblast (MEF) feeders and leukemia inhibitory factor (LIF). Hanging drops provide a uniform aggregate size, which is then expanded by continued growth in suspension culture. The embryoid bodies are then plated and allowed to differentiate further in culture. - [Read Differentiation of Embryonic Stem (ES) Cells Using the Hanging Drop Method]

Category: ELISA Protocols

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. - [Read ELISA Method to Measure Inhibition of the COX Enzymes Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

The effect of a test compound, in the presence and absence of S-9 mix, on the differentiation and growth of rat limb bud and CNS cells in vitro indicates whether it is potentially a teratogen in vivo. - [Read In Vitro Micromass Teratogen Assay]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract.  A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

Category: Drosophila Protocols

Method relies on the examination of the ear’s mechanics, which is actively modulated by the motility of auditory neurons and reflects the function of mechanosensory proteins these cells comprise [5-7]. Mechanical signatures arising from the motility of the neurons are assayed by measuring the vibrations of the antennal sound receiver in the presence and absence of sound. - [Read Mechanical Tracing of Protein Function in the Drosophila Ear Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields low background absorbance values in the absence of cells. Includes: Determining optimal cell counts, performing an assay, data interpretations and troubleshooting. - [Read MTT Cell Proliferation Assay Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to test whether a transcription factor disrupts the chromatin of a promoter of a gene of interest. First, chromatin is assembled in vitro on the gene of interest in the presence and absence of a transcriptional activator (see Protocol on Assembly of Chromatin with Drosophila S-190 Chromatin Assembly Extract and Transcriptional Activators). - [Read Nucleosomal Array Disruption Assay Protocol]

Category: Cytoskeleton Protocols: Microtubules Protocols

Recycle tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day use. Generally store recycled tubulin in Injection Buffer (IB) without free GTP. This is done because depolymerization appears to be much better in IB, IB is ideal for microinjections/adding tubulin to extracts, and the absence of free GTP makes polymerization with GMPCPP, a very useful GTP analog that has ~5-10X lower affinity than GTP for tubulin. - [Read Recycling Tubulin Protocol]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Category: Transfection Protocols

Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids. This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides. The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate. - [Read Transfection of Mammalian Cells with siRNA Duplexes Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids.  This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides.  The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate. - [Read Transfection of Mammalian Cells with siRNA Duplexes Protocol]