primary Protocols

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

The results of cytotoxicity tests in primary cultures of rat hepatocytes and in MDBK and McCoy cells can be used to predict the in vivo 4-wk maximum tolerated dose in rats and dogs.  A correlation between in vitro cytotoxicity, as measured in this system, and LD50 values in rats and mice has also been established. - [Read In Vitro Prediction of the Maximum Tolerated Dose Protocol]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

This protocol describes the isolation of fibroblasts from mouse embryos. Mouse embryonic fibroblast (MEF) cells are used as a feeder layer for the culture of mouse embryonic stem (ES) cells to help maintain them as pluripotent stem cells. The inhibition of ES-cell differentiation provided by the MEF feeders appears to be due to their production of leukemia inhibitory factor (LIF). - [Read Isolation and Freezing of Primary Mouse Embryonic Fibroblasts (MEF) For Feeder Plates]

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: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

Embryonic stem cells are usually grown on a layer of mitotically inactivated primary mouse embryonic fibroblasts to promote growth and prevent differentiation. Boris Greber - [Read Isolation and handling of primary mouse embryonic fibroblasts (MEFs)]

Category: Cell Biology and Cell Culture: Epithelial Cell Culture

Isolation of Primary Epithelial Cells. Digestion: In Collagenase Medium, Purification and epithelial cell culture methods. Bissell Lab. - [Read Isolation of Primary Epithelial Cells]

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

Isolation of Primary Fibroblasts from Mouse Embryos. Bradley's Lab, Texas. - [Read Isolation of Primary Fibroblasts from Mouse Embryos]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

Isolation of Primary Mouse Embryonic Fibroblasts (PMEFs). ESGRO. - [Read Isolation of Primary Mouse Embryonic Fibroblasts (PMEFs)]

Category: Antibody Protocols: Antibody Labeling Protocols

This protocol describes the covalent coupling of antibodies to biotin. Biotin groups bind with extremely high affinity to streptavidin and avidin, both of which are available commercially coupled with enzymes, fluorescent dyes, or iodine. A biotinylated primary antibody, therefore, can be detected with any of a wide variety of different labels. The biotinylation reaction is simple and mild, and rarely inactivates the antibody. - [Read Labeling Antibodies with Biotin Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

Direct labeling of purified antibodies is the method of choice when simultaneously visualizing two or more antibodies of the same species, class, or subclass. This allows the localization of multiple antigens to be compared in the same cell, tissue, or sample. Labeled primary antibodies are also useful for improving background-to-readout ratios, and they can be essential for immunoassays in which good quantification is needed. - [Read Labeling Antibodies with Fluorochromes Protocol]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Confocal laser scanning microscopy (CLSM) is a relatively new light microscopical imaging technique which has found wide applications in the biological sciences. The primary value of the CLSM to the biologist is its ability to produce optical sections through a 3-D specimen-e.g., an entire cell or a piece of tissue - that, to a good approximation, contain information from only one focal plane. Article includes principle and applications of confocal laser scanning microscope. - [Read Looking Inside Cells and Tissues by Optical Sectioning with a Confocal Laser Scanning Microscope]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Mammalian toxicology for acute toxicity studies.  Includes: M101 Acute Limit Test (Rodents), M102 Acute Toxicity Study (Rats or Mice), M120 Dose Escalation Study (Dog), M103 Acute Dermal Limit Test, M104 Acute Dermal (LD50), Study M105 Primary Dermal Irritation, M106 Primary Eye Irritation Study, M106-A, M110 Local Lymph Node Assay in Mouse, M115 Rabbit Vaginal Irritation Assay. - [Read Mammalian Toxicology Acute Toxicity Studies]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Protocol describes, samples containing the target protein are deposited onto a polyvinyldifluoride (PVDF) membrane using a vacuum manifold.  The immobilized protein is exposed to an antibody specific for the target protein, followed by an antibody that reacts with species-specific determinants carried by the primary antibody and is conjugated to horseradish peroxidase (HRP). - [Read Measuring Protein Concentration by Western Analysis Using Enhanced Chemiluminescence Detection]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

Preparation of primary mouse embryonic fibroblasts, Preparation of mitotically inactive mouse embryo fibroblasts, Irradiation of the MEFs. Nederlands Kanker Instituut Teriele. - [Read MOUSE EMBRYONIC FIBROBLASTS PDF]

Category: Immunology: T Cell Protocols

Protocol for nucleofection and adoptive transfer of primary mouse T lymphocytes. - [Read Nucleofection and Adoptive Transfer of Primary Mouse T Lymphocytes Protocol]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection of genes into naïve mouse T cells using nucleofection using a modified electroporation technique. This protocol offers the possibility for rapid functional in vivo studies of targeted genes in primary mouse T cells. - [Read Nucleofection and Adoptive Transfer of Primary Mouse T lymphocytes using Transient Transfection]

Category: PCR Protocols

PCR can be used to identify rare DNA sequences in DNA libraries by increasing the abundance of a particular sequence.  This is accomplished by subdividing the original library into pools of decreased complexity and screening each pool or group of pools for a given DNA sequence.  A pool that contains the desired clone is subsequently subdivided into smaller pools, each of which is screened using the same PCR protocol that was used for the primary screen. - [Read PCR-Based Screening of DNA Libraries Protocol]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

Protocol for the preparation of feeder layers from primary embryonic fibroblasts. - [Read Preparation of Feeder Layers from Primary Embryonic Fibroblasts Protocol]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

MEF feeders are prepared weekly to provide a substrate for undifferentiated embryonic stem (ES) cells. Primary MEF cells are thawed, established in culture, treated with mitomycin C to halt their proliferation so they cannot overgrow the ES cultures, and then replated onto dishes convenient for ES cell culture. This protocol can also be used to prepare feeder cells from STO fibroblast cell lines. - [Read Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol]

Category: Cell Biology and Cell Culture: Hepatocyte Cell Culture

USC Liver Transplant Surgery Program and Center for Liver Disease. Preparation of primary cultures of rat hepatocytes (or freshly isolated hepatocytes). - [Read Preparation of primary cultures of rat hepatocytes (or freshly isolated hepatocytes)]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Protocol for primary culture of rat cerebral astrocytes. - [Read Primary Culture of Rat Cerebral Astrocytes Protocol]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

This protocol describes the culture of embryonic stem (ES) cells using mitotically inactivating primary mouse embryonic fibroblast (MEF) cells as a feeder layer (preparation described in Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates). The ES culture medium is supplemented with recombinant leukemia inhibitory factor (LIF) to help maintain the cells as pluripotent stem cells. This protocol has been optimized for the ES-D3 cell line. - [Read Propagation of Pluripotent Mouse Embryonic Stem (ES) Cells Protocol]

Category: Stem Cell Protocols: Mouse Embryonic Fibroblasts Protocols

This protocol describes the culture of embryonic stem (ES) cells using mitotically inactivating primary mouse embryonic fibroblast (MEF) cells as a feeder layer (preparation described in Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates). The ES culture medium is supplemented with recombinant leukemia inhibitory factor (LIF) to help maintain the cells as pluripotent stem cells. This protocol has been optimized for the ES-D3 cell line. - [Read Propagation of Pluripotent Mouse Embryonic Stem (ES) Cells Protocol]

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Protocol for primary cultures of HUVECs. Includes: Buffers and reagents (Fetal calf serum, Phosphate Buffer Saline (PBS), Collagenase, Buffer for conservation and transport of umbilical cords, Culture medium); Cell culture. - [Read Protocol for Primary Cultures of HUVECs]

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

Protocol for primary cultures of HUVECs. - [Read Protocol for Primary Cultures of HUVECs]

Category: Protein Protocols: Protein Electrophoresis: SDS-PAGE Electrophoresis Protocols

PROTOCOL FOR SDS-PAGE. Immuno-Physiology Laboratory, Florida International University. Includes: Primary considerations, preparing samples, loading samples, running gels, and quantitative staining. - [Read PROTOCOL FOR SDS-PAGE]