study Protocols

Category: Genetics and Genomics: Cancer Genetics Protocols

Anatomy of a comparative gene expression study. Includes: Choosing Cell Populations; mRNA Extraction and Reverse Transcription; Fluorescent Labeling of cDNA's; Hybridization to a DNA Microarray; Scanning the Hybridized Array; Interpreting the Scanned Image. - [Read Anatomy of a Comparative Gene Expression Study]

Category: Microarray Protocols: Microarray Analysis Protocols

Information on anatomy of a comparative gene expression study.  Includes: Choosing cell populations; mRNA Extraction and Reverse Transcription; Fluorescent labeling of cDNA's; Hybridization of DNA microarray; Scanning the hybridized array; Interpreting the scanned image. - [Read Anatomy of a Comparative Gene Expression Study]

Category: DNA Protocols: DNA Extraction Protocols: Extracting Ancient DNA

The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, but PCR can be used to amplify and study short mitochondrial DNA sequences thatare of anthropological and evolutionary significance. SVANTE PAABO, PNAS. - [Read Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. PDF]

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]

Category: Signalling Signal Transduction Protocols: Chemokine Protocols

Simple apparatus to study chemotaxis of leukocytes or other migratory cells. Protocol for Chemotaxis. BioProtocol - [Read Chemotaxis Assay Using Microchemotaxis Chambers (Modified Boyden Chamber)]

Category: Reporter Gene Assays: Luciferase Assay Protocols

It is possible that some cell lines have lost the ability to perform RNAi or that cells derived from certain tissues do not support RNAi. This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: Cell Culture Protocols

Protocol used to study secretion of proteins and prostaglandins by endometrium from the cow, ewe, mare, bitch and other species. The technique is also useful for culture of peri-implantation conceptuses and placental tissues for metabolic labelling studies and to obtain conceptus secretory proteins for biological studies.The medium used is called Pig MEM, which is a modified minimum essential medium supplemented with non-essential amino acids, vitamins, insulin and additional glucose. - [Read Culture of Endometrial Explants and Peri-implantation Conceptuses to Monitor Synthesis and Secretion]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Culture conditions have been established for a second blastocyst-derived cell line, trophoblast stem (TS) cells, in addition to embryonic stem (ES) cells. This protocol describes a method for culturing TS cell lines. These cells can then be used to study trophoblast differentiation and placental function. - [Read Culturing Trophoblast Stem (TS) Cell Lines Protocol]

Category: Microscopy Protocols: Electron Microscopy Protocols

Article describe the preparation of cells for correlative electron microscopy after live light microscopic observation of fluorescently labeled cytoskeletal proteins microinjected into the same cells. Since identification of cytoskeletal elements in electron microscopic preparations is an essential part of any correlative study, procedures for immunogold labeling of cytoskeletal components and for myosin S1 decoration of actin filaments are also described. - [Read Electron Microscopy of the Cytoskeleton of Cultured Cells]

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

Protocol describes how monocytes can be sorted from a lysed whole blood sample, and that cytospin preparations can be made for examination by light microscopy. - [Read Enrichment Of Monocytes For Morphological Study Protocol]

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

Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. - [Read Flow Cytometry of Apoptosis Protocol]

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

This protocol is concerned with the use of iodixanol gradients in an analytical mode to study the membrane localization of a particular protein or function. Continuous gradients are best suited to this task. One of the protocols described in this protocol starts with a discontinuous gradient, but since the gradient is centrifuged at 174,000g for 16 h it will become continuous by diffusion. - [Read Fractionation of Yeast Membranes in Pre-Formed Continuous Iodixanol Gradients]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

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

This method enables the culturing of thyroid cells without loss of differentiation and medium change. It is potentially useful for the long-term study of drug effects on the thyroid gland. - [Read Human Thyroid Culture Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Protocol for immunostaining of DDR proteins in senescent cells. Detailed methodology to study the activation of a number of DNA damage response factors in senescent human fibroblasts. - [Read Immunostaining of DDR Proteins in Senescent Cells Protocol]

Category: Immunology: Immunofluorescence Protocols

The study of the activation of DNA damage response factors at the single cell level relies on the observation of their accumulation (or the accumulation of their activated forms) in nuclear foci by plain immunofluorescence techniques. - [Read Indirect Immunofluorescence with Preextraction (In Situ Cell Fractionation) Protocol]

Category: Immunology: T Cell Protocols

Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

Category: Mouse Protocols: Mouse Embryology Protocols

Isolation of extraembryonic tissues allows one to study normal mouse development as well as the molecular basis of defects which cause fetal death. This protocol describes a method for isolating extraembryonic membranes from pregnant mice. - [Read Isolating Mice Extraembryonic Membranes Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause postimplantation defects. This protocol describes a method for isolation of early neural-fold-stage embryos. - [Read Isolating Postimplantation Embryos: Early Neural-Fold-Stage Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause post-implantation defects. This protocol describes a method for isolating early primitive-streak-stage embryos. - [Read Isolating Postimplantation Embryos: Early Primitive-Streak-Stage Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause postimplantation defects. This protocol describes a method for isolating early somite-stage embryos (~8.5 days post coitum [dpc]). - [Read Isolating Postimplantation Embryos: Early Somite-Stage Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause postimplantation defects. This protocol describes a method for isolating late primitive-streak-stage embryos (~7.5 days post coitum [dpc]). - [Read Isolating Postimplantation Embryos: Late Primitive-Streak-Stage Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations that cause post-implantation defects. This protocol describes a method for isolating prestreak-stage embryos (~5.5 days post coitum [dpc]). - [Read Isolating Postimplantation Embryos: Prestreak-Stage Protocol]