parameters Protocols

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. - [Read Calibration of Micropipette Tips Protocols]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Basic information on confocal microscopy, includes: Specimen Preparation and Imaging; Objective Lens Parameters and Optical Section Thickness; The Objective Lens; Probes for Confocal Imaging; Autofluorescence; Collecting Images; Troubleshooting; Image Processing and Publication; - [Read Confocal Microscopy: Speciman Preparation and Imaging]

Category: Transfection Protocols

Protocol should be viewed as a starting point for systematic optimization of transfection mediated by lipofecting agents. Once a positive signal has been obtained from a transfected plasmid carrying a standard reporter gene, optimal conditions for transfection can be established by systematic variation of parameters such as the initial cell density, the amount and purity of DNA, the media and serum, and the time of exposure of the cells to the cationic-lipid-DNA complex. - [Read DNA Transfection Mediated by Lipofection Protocol]

Category: Immunology: B Cell Protocols

B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. Provides methods for the latter (direct) approach--namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II-antigen expression. - [Read Early Events in B Lymphocyte Activation Protocol]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

Information for EMSA (Gel Shift) technique. Includes: Critical EMSA Reaction Parameters. - [Read EMSA (Gel Shift) Technique Information]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Provides information on specific parameters you need to be aware of when using eukaryotic in vitro translation systems. Includes: DNA Template Considerations; Protein Labeling; Non-Radioactive Protein Labeling. - [Read Eukaryotic In Vitro Translation Systems]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Parameters for fixation and staining. Includes: Antigen, Fixation, Fixation time, Antibody and Dilution. - [Read Fixation and Staining Parameters]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

Generally in iodixanol gradients the density of organelles decreases in the series: peroxisomes, mitochondria, lysosomes, ER, Golgi, although in Dictyostelium discoideum, the lysosomes are denser than the mitochondria. Iodixanol gradients can usually provide satisfactory resolution of all these membrane particles although it may be necessary to modulate either the gradient or centrifugation parameters in order to optimize a particular separation. - [Read Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

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

The basis of this procedure is that two specific cell type preparations may be isolated, exposed separately to various compounds over a range of concentrations, and the cytotoxicity of these determined.  Parameters deemed indicative of a cytotoxic effect include a reduction in de novo protein synthesis and decreased glucose and fatty acid metabolism.  A cytotoxic effect may indicate that a chemical is likely to be nephrotoxic in vivo. - [Read Isolated Rat Glomeruli and Proximal Tubules]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope when three planes along the z-axis of the cell are acquired. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP & Bright Field—Three Z Axis]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP and Bright Field Images]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Movie Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP Time Series for Markers]

Category: Primary Cell Isolation and Culture Protocols

There are several manual methods that can be used to perform tissue microdissection. Techniques using hand-held tools as well as mechanical micromanipulator-based approaches have been described. However, speed and precision are the most important parameters and any method that achieves these is adequate. Investigators should also expect to invest time initially by practicing on 10 to 20 cases to begin to feel comfortable with the technique. - [Read Manual Microdissection]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions. - [Read Nucleic Acid Hybridization General Aspects]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition of confocal fluorescent and bright field images of live cells, expressing cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing; Movie Processing. - [Read Protocol for Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells on a Zeiss]

Category: Real Time PCR

Real-Time Chemistry, Instrumentation, Real-Time PCR Quantitation, Quantitative Primer and Probe Design, Thermal Cycling Parameters, Sample Preparation, and Real-time Data Analysis. - [Read Real-Time or Kinetic PCR]

Category: Real Time PCR

Learn Quantitative PCR Methodology. Assay parameters, Melt Curves and Primer Dimers, Standard Curves, Dynamic Range, Quantification methods, Delta CT, Standard Curves. Dr.Powell. - [Read Real-Time PCR (qPCR) Basics]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes transfection of plasmid DNA into primary hippocampal neurons using DNA/calcium-phosphate (CaPO4) coprecipitation. The precise pH of the transfection medium and the incubation time of cells with the coprecipitate are critical for reproducible and efficient transfection. Once these parameters are optimized for a given plasmid, the method is easily adapted for transfection of other established cell lines. - [Read Transfection of Hippocampal Neurons with Plasmid DNA Using Calcium Phosphate Coprecipitation]