human Protocols

Category: Mouse Protocols: Mouse Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Isolation and Growth of Primary Cell Cultures from Mouse Protocol]

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

Methodological details about sorting and purification of peripheral blood and decidual human NK subsets. - [Read Isolation of Human NK Subsets Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

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

Potential embryotoxicity is assessed by monitoring the effect of the test compound on total protein synthesis, and DNA synthesis in cultured human foetal lung fibroblasts.  Rat lung epithelial cells can be used to determine cytotoxicity of select compounds because of their ability to metabolise xenobiotics. - [Read Lung Cell Assay Protocol]

Category: Fungi Protocols: Aspergillus Protocols

Gliotoxin is a metabolite of Aspergillus fumigatus that exhibits immunosuppressive activity against certain cells of the immune system. Secretion of gliotoxin during infection has been suggested as being a factor in the pathogenesis of aspergillosis. Gliotoxin secretion can be assayed in a number of ways by thin layer chromatography (TLC) high performance liquid chromatography (HPLC) or bioassay using the effect of gliotoxin on human cells1. - [Read Method for Assaying Gliotoxin Production in Aspergillus fumigatus Protocol]

Category: Microarray Protocols: Microarray Analysis Protocols

Description of gene array analysis and normalization - [Read Microarray Analysis of Highly Purified Human Decidual NK Cells Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Human tissues are comprised of multiple interacting cell populations in a complex three dimensional arrangement with each cellular phenotype determined by a unique profile of mRNA and protein expression. Before microdissection techniques were developed, the only analysis tools for phenotypic studies were primarily immunohistochemistry and in-situ hybridization. While useful, these tools are limited to single gene analysis and, in general, do not allow qualitative studies. - [Read Microdissection Overview]

Category: Cytogenetics Protocols

MN in Human Lymphocytes (method and protocol description) Nina T. Holland, Division of Environmental Health Sciences, Berkeley - [Read Micronucleus assay Human Lymphocytes]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Molecular and genetic toxicology studies for in vitro cytogenetics with CHO or human lymphocytes. Includes: G401 Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells, G401-2, G401-R, G401-R2, G401-R2H, G402 Chromosomal Aberrations in Human Lymphocytes, G402-2, G402-R, G402-R2, G402-R2H, G403 Sister Chromatid Exchanges in Chinese Hamster Ovary (CHO) Cells. - [Read Molecular and Genetic Toxicology Studies for In Vitro Cytogenetics with CHO or human lymphocytes]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol for oligofection of small interfering RNAs in senescent human fibroblasts. This protocol allows the transfection of human senescent cells (regardless of the cause of cellular senescence) with an efficiency of around 70%. - [Read Oligofection of Small Interfering RNAs in Senescent Human Fibroblasts Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Primer pairs will amplify sequences present as a single copy in the mouse genome with the Universal Genotyping Protocol. Includes: b-Galactosidase (LacZ); cre-recombinase; CFP; diphtheria toxin; dsRED; Fabpi-200; Fabpi-500; flp recombinase; GFP/BFP/YFP; human growth hormone (complete); human growth hormone (transcriptional stop); luciferase (click-beetle); luciferase (firefly); neomycin phosphotransferase; SRY (male-specific); tTA (tet-on). - [Read PCR Genotyping Primer Pairs Protocols]

Category: Nucleic Acid Detection Protocols

Protocol for preparation of fluorescent DNA probe from HUMAN mRNA or total RNA using direct incorporation. Includes: Preparing fluoresenctly labeled cDNA (probe); Washing and Scanning Arrays. - [Read Preparation of Fluorescent DNA Probe from HUMAN mRNA or Total RNA using Direct Incorporation]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol for the preparation of fluorescent DNA probe from HUMAN mRNA or total RNA using direct incorporation. Includes: Preparing fluoresenctly labeled cDNA (probe); Washing and Scanning Arrays. - [Read Preparation of Fluorescent DNA Probe from HUMAN mRNA/ Total RNA using Direct Incorporation Protocol]

Category: Fungi Protocols

Protocol for the preparation of solid tissue for Aspergillus galactomannan antigen detection by Platelia (Biorad). Technique was designed for use on human serum. However, it may also be possible to perform this method on solid tissues and organic solutions. Viscous solution and tissue specimens need to be pre-treated to achieve the extraction of the Aspergillus antigen and to get a homogeneous sample in solution. - [Read Preparation of Solid Tissue for Aspergillus Galactomannan Antigen Detection by Platelia Protocol]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

Includes protocols: Mouse Embryonic Fibroblasts (MEF) Preparation; Harvesting MEFs; Cryopreservation of MEFs; Thawing and maintaining MEFs; Irradiating & Plating MEFs; Culture of Human ES cells with Matrigel® and Conditioned Medium; Preparation of Conditioned Medium (CM); Preparation of Matrigel® -coated plates; Passage of human ES cells on Matrigel®; Daily maintenance of feeder-free culture; Freezing Human ES Cells; Thawing Human ES cells; Formation of Embryoid Bodies; - [Read Protocols for the Maintenance of Human Embryonic Stem Cells in Feeder Free Conditions]

Category: Cytoskeleton Protocols: Intermediate Filaments Protocols

Intermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes a method for purifying one type of IF, vimentin, from bovine lens tissue. Purification of human vimentin expressed in Escherichia coli is also described. These methods are useful in the preparation of other IF protein subunits for microinjection studies as well. - [Read Purification of Bovine Lens and Bacterially Expressed Human Vimentin Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

Protocol for the purification of human mononuclear cells and neutrophils. - [Read Purification of Human Mononuclear Cells and Neutrophils Protocol]

Category: Immunology: T Cell Protocols

In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2. - [Read Quantitation of Functional T Cells by Limiting Dilution Protocols]

Category: Radioactivity: Radioaction Safety

Administration of the Radiation Safety Program, , Physical Security of Radioactive Materials, "As Low as Reasonably Achievable" (ALARA) Philosophy, Authorized Users, Radiation Workers , Permitted Workers, Training, Procurement, Inventory and Records, Personnel Monitoring, Radiation Exposure During Pregnancy, Laboratory Supplies and Equipment, Bioassay, Labeling, Surveys, Radioactive Waste Disposal. U.S. Department of Health and Human Services, CDC, Atlanta, GA. - [Read Radiation Safety Manual]

Category: Animal Research Techniques

Handling Methods for animals: mice, rats, hamsters, rabbits, cats, dogs, non-human primates, swine, goats, sheep, calves, pigs. Restraint devices, drug administration. - [Read Restraint and Handling of Animals Guide]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the retroviral infection of primary human keratinocytes (PHKs). - [Read Retroviral Infection of Primary Human Keratinocytes (PHKs) Protocol]

Category: Cell Biology and Cell Culture

Method separates monocytes and lymphocytes on the basis of density and size. - [Read Separation of monocytes from human leukocyte-rich plasma by flotation.]

Category: Cell Biology and Cell Culture

This protocol describes how to isolate human platelets from whole blood. Isolated platelets are used for static adhesion assays, for flow chamber assays, flow cytometry measurements, etc. - [Read Separation of Platelets from Whole Blood]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: ES Cell Growth Media and Requirements

Human embryonic stem cells are a valuable resource for research and cell replacement therapy but are notoriously cumbersome to culture. Bhatia and colleagues show that an increased dose of basic fibroblast growth factor eliminates the need for feeder laye - [Read Simplifying hESC culture]

Category: Genetics and Genomics: Genotyping Protocols

Describe the current efforts to identify and characterize the large number of SNP's required and discuss the practicalities of association studies for the identification of genes involved in complex traits. - [Read Single Nucleotide Polymorphisms as Tools in Human Genetics]