normal Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol provides a method for analysis of DNA fragmentation using flow cytometry after propidium iodide (PI) labeling of apoptotic nuclei. Apoptotic nuclei stain less than their normal counterparts because of diffusion of low-molecular-weight fragments out of the cells and/or chromatin condensation. - [Read Analysis of DNA Fragmentation Using Propidium Iodide (PI) Fluorescence of Individual Nuclei Protocol]

Category: Cytogenetics Protocols

This CGH Protocol is used for DNA of good quality when available in sufficient amounts. We usually do replicate hybridizations using samples labeled "inversely" (reversing the label for test and normal DNA's). If appreciable artifact occurs, then alternative labels are tried. - [Read CGH of Direct Labeled Test DNA vs Normal DNA Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

The starting material for de novo isolation of stem cell lines can be either normal 3.5-days post coitum (dpc) expanded blastocysts or "delayed" blastocysts. Delayed blastocysts are usually collected 4-6 days after ovariectomy. For both groups of blastocysts, tissue culture procedures are similar. The only difference is the timing of the first disaggregation, because delayed blastocysts will initially grow more slowly. - [Read De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes. Protocol was optimized from a protocol using 35S-labeled RNA probes. It allows to detect the expression of low abundant mRNAs in the cardiovascular system, e.g. of the proinflammatory cytokine GM-CSF in normal human coronary arteries, and of IL6 and gp130 in human failing hearts. The protocol can be combined with immunohistochemistry. - [Read Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. As the cells age, so their density tends to increase; reticulocytes therefore tend to have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite varied, from quite broad ones. - [Read Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol]

Category: Cell Culture Protocols: Cell Immortalization Protocols

Cultivating animal cells in the laboratory is an indispensable technique for cell biologists. However, most normal primary cell lines, while faithfully reproducing the phenotype of their tissue of origin, do not grow indefinitely in culture. After a series of population doublings (the number of which varies by species, cell type, and culture conditions) primary cells enter a state where they no longer divide. - [Read Immortalization of Cells in Culture]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Category: Yeast Protocols

Protocol for in vitro endoplasmic reticulum to golgi transport reaction in Yeast. Includes: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast Protocol]

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]

Category: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes a method for Caesarean section and fostering. Caesarean section is required if the recipient of an embryo transfer or any pregnant mouse has not given birth by the delivery time normal for the particular strain. - [Read Mouse Caesarean Section and Fostering Protocol]

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

Protocol includes information on: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read Protcol for In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast]

Category: Protein Protocols: Protein Precipitation Procols

TCA-DOC Normal, TCA Acetone Precipitation, Ethanol Precipitation, TCA-DOC / Acetone, Acidified Acetone/Methanol, Dr. Mario Lebendiker. University of Jerusalem. - [Read Protein Precipitation Protocols]

Category: Cell Biology and Cell Culture

Protocol for the recovery of a highly viable fraction for use in fertilization. Protocol uses an iodinated density gradient medium to adjust the density of the raw ejaculate to approx 1.170 g/ml by mixing with a high density medium (ρ >1.26 g/ml) and loading it beneath a discontinuous gradient. - [Read Purification of viable bovine spermatozoa of normal morphology.]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when slightly shaken and lymphocytes will not cluster together as much as normal. If cultures are suspect, a drop of culture can be streaked on a YPD media plate to check for growth of yeast colonies, or a 5 ml sample can be taken to Barnes Diagnostic Center for identification of yeast strain. - [Read Removal of Yeast Contamination from Lymphoblast Cultures Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol for sizing of YAC clones. To determine the size of a yeast artificial chromosome within the background of the normal yeast chromosomal complement. - [Read Sizing of YAC Clones Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Microorganism Cytotoxicity Assays Protocols

The basis of this test is that a cytotoxic chemical (regardless of site or mechanism of action) will interfere with the normal motility of the protozoan, Tetrahymena thermophila, in culture.  The degree of interference of motility as compared to control cultures, related to the concentration of the test compound, provides an indication of toxicity. - [Read Tetrahymena Thermophila Ocular Irritancy Test]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

A. tumefaciens is a soil-dwelling bacterium that transforms normal plant cells into tumor-forming cells by inserting a piece of bacterial DNA (the transfer, or "T," DNA) into the plant cell genome. The Ti plasmid also carries many of the transfer functions for mobilizing the T-DNA. This article provides a brief discussion of the principles of T-DNA transformation, including consideration of T-DNA vectors and their hosts. - [Read Vectors and Agrobacterium Hosts for Arabidopsis Transformation Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

During development many plant cells undergo endoreduplication, whereby ploidy increases to a multiple of the normal 2C content. For eg., trichome development is accompanied by an increase in ploidy to 32C, indicating that trichome cells undergo four rounds of endoreduplication. Protocol describes DNA levels, and hence developmental progress in the corresponding cells, are measured by staining the DNA with a fluorescent marker and then quantifying the fluorescence of individual nuclei. - [Read Whole-Mount DAPI Staining and Measurement of DNA Content in Plant Cells]