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Assay of Intracellular Free Calcium in Suspended B Cells PDF - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi?pid=PP00000011

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]

Assay of Intracellular Free Calcium in Suspended B Cells Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000011.pdf?pid=PP00000011

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]

Assay of Intracellular Free Calcium in Suspended B Cells Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000011.pdf?pid=PP00000011

Category: Immunology: B Cell Protocols

Assays for B Lymphocyte Function Protocols - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E66340AD6485AA0FEB8F538B2FD389E&objectid=6674A538AC9B642C5CD51556EF7B3D26

Category: Immunology: B Cell Protocols

Describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. - [Read Assays for B Lymphocyte Function Protocols]

Assembling Aggregates between Embryonic Stem (ES) Cells and Diploid Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4424

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for assembling aggregates between ES cells and diploid embryos. The resulting chimeras are useful for separating certain extraembryonic phenotypes from phenotypes in the embryo proper, since the diploid embryo contributes to all parts of the conceptus, but the ES cell component does not contribute to the trophoblast or yolk sac endoderm. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Diploid Embryos Protocol]

Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4426

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing ES cell-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for ES cell-diploid embryo aggregation in which diploid embryos are replaced with tetraploid embryos. The resulting chimeras can be used to analyze the embryonic versus extraembryonic phenotype of a mutation. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos Protocol]

Attaching Suspension Cells to Slides Using a Cytocentrifuge Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4291

Category: Cell Culture Protocols: Cell Staining Protocols

A simple method for handling suspension cells is to attach them to a solid substrate before fixation, and one way to do this is to use a cytocentrifuge. - [Read Attaching Suspension Cells to Slides Using a Cytocentrifuge Protocol]

B- Galactosidase Assay Protocol - http://www.biochem.northwestern.edu/ibis/morimoto/research/Protocols/VI.%20Transcription/I.%20b-gal%20.pdf

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The recommended amount of RSV-ß-Galactosidase plasmid to use for transfection of cells (60 mm or 100 mm dish) is 1-2 µg. The optimal amount of plasmid DNA will be determined by the efficiency of transfection , which is very dependent upon the particular cell line and transfection protocol. - [Read B- Galactosidase Assay Protocol]

Bacterial Transformation Electroporation Protocol - http://rothlab.ucdavis.edu/protocols/T10.html

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol for bacterial transformation electroporation. Includes: Preparation of Electrocompetent cells. - [Read Bacterial Transformation Electroporation Protocol]

Baculovirus Growing and Freezing Insect Cells Protocol - http://www.ebioscience.com/ebioscience/appls/BV.htm#C

Category: Cloning Protocols: Baculovirus Protein Expression Protocols

Protocol for Baculovirus growing and freezing insect cells. - [Read Baculovirus Growing and Freezing Insect Cells Protocol]

Baculovirus Protein Expression Protocols - http://www.ebioscience.com/ebioscience/appls/BV.htm

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Baculovirus protein expression protocols. Includes protocols: Cotransfection Protocol; Analysis of nonsecreted protein expression; Growing and Freezing Insect Cells. - [Read Baculovirus Protein Expression Protocols]

Basic Techniques for Mammalian Cell Tissue Culture Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E662B5FE053DEE8CB4AC3EB6F729A11&objectid=667396C6F0D96C40EB68A3D8DB8B201A

Category: Cell Culture Protocols: Sterile Technique Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Beta b-Galactosidase Color Assay for Cultured Cells - http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/betagal.txt

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Beta b-Galactosidase Color Assay for Cultured Cells. Bradley's Lab, Texas. - [Read Beta b-Galactosidase Color Assay for Cultured Cells]

Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4450

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

C. elegans RNAi Protocol - http://www.zoology.ubc.ca/~alorch/rnai-protocol.htm

Category: C. elegans Protocols: C. elegans Genetics Protocols

Protocol for C. elegans RNAi. Includes: Transformation of competent cells; Blunt-end ligation; Preparation of competent cells; Dephosphorylation of linear plasmid DNA; Restriction Digest: EcoRV; Insert amplification from gDNA; Gel purification: QiaQuick gel purification kit; Mini-prep; Transformant Screening; Bacterial preparation and induction; Preparation of worms for RNAi feeding. - [Read C. elegans RNAi Protocol]

C2 Preparation of pre-formed iodixanol gradients for mammalian cells. - http://www.axis-shield.com/densityhome/optiprep/C02.pdf

Category: Cell Biology and Cell Culture

Techniques on how to create gradients of iodixanol for the fractionation of mammalian cells. These gradients can be generated as pre-formed discontinuous or continuous gradients. These gradients are invariably run in swinging-bucket rotors in low-speed centrifuges. - [Read C2 Preparation of pre-formed iodixanol gradients for mammalian cells.]

C8 Isolation of bovine peripheral blood mononuclear cells by flotation. - http://www.axis-shield.com/densityhome/optiprep/C08.pdf

Category: Cell Biology and Cell Culture

The protocol presented in this Application Sheet uses an alternative strategy to sedimentation on to a density barrier, that is to adjust the density of whole blood to a value just greater than the cells of interest and allow them to float to the surface. - [Read C8 Isolation of bovine peripheral blood mononuclear cells by flotation.]

CaCl2 Competent Cell Stock Protocol - http://www.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Simple Protocol for CaCl2 Competent Cells. Chen Lab. Indiana University Bloomington - [Read CaCl2 Competent Cell Stock Protocol]

CALCIUM PHOSPATE TRANSFECTION OF HUMAN CELLS - http://omrf.ouhsc.edu/~frank/CAPO4TRANSFECT.html

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Simple Transfection using Calcium chloride and phosphate.Dr. Frank. Arthritis and Immunology Program, Oklahoma City. Medical Research Foundation. - [Read CALCIUM PHOSPATE TRANSFECTION OF HUMAN CELLS]

Calcium phosphate Tranfection for MDCK cells - http://www.its.caltech.edu/%7Ebjorker/Ca-P_Transfection_MDCK.pdf

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Protocol for cell transfection, clone screening and picking cell clones. Bjorkman Group. California I.T - [Read Calcium phosphate Tranfection for MDCK cells]

Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3872

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol,is based on the venerable method of Graham and van der Eb (1973). The procedure is used chiefly to generate stable lines of cells carrying chromosomally integrated copies of the transfected DNA. - [Read Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA]

Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3871

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

Calcium phosphate forms an insoluble precipitate with DNA, which attaches to the cell surface and is taken into the cells by endocytosis. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent. This protocol is a modified version of a method published by Jordan et al. (1996) who rigorously optimized calcium-phosphate-based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells. - [Read Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs]

Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements - http://cyto.mednet.ucla.edu/Protocols/relative.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Calibration Protocols

Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels. - [Read Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements]

Calibration of Micropipette Tips Protocols - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4655

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]

Articles (1-10 of 24)

Lambda Phage Protocol Large DNA Preparation

Single Stranded Plasmid DNA Isolation Protocol

A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

Microinjection Pipettes Protocol

Microinjection Pipettes Preparation Protocol.

Acid Washing Microinjection Pipettes Protocol

Acid Washing of Glass microinjection pipettes protocol.

Paraffin Embedding Protocol

Paraffin Embedding Protocol for molecular profiling. This Paraffin Embedding Protocol describes the processing of the tissues into sections following ethanol fixation. Molecular profiling (MP) is a technique that is used to visualize the global patterns of RNA expression or protein expression in various cell types and disease processes.

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