cells Protocols

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages. - [Read (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel]

Category: Immunology: Chemotaxis

12-well Chemotaxis Chamber Protocol. Preparing the Chamber, Preparing and Adding Responding Cells, Removing, Wiping and Staining the Filter. Neuroprobe - [Read 12-well Chemotaxis Chamber Protocol]

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

The incorporation of 5-bromo-2-deoxyuridine (BrdU) in replicating DNA of proliferating cells can be used to measure their proliferation. This protocol allows the identification of cells which have incorporated BrdU by flow cytometry. - [Read 5-Bromo-2-Deoxyuridine (BrdU) and 7-Amino-Actinomycin (7-AAD) Staining for Cell Proliferation Assay]

Category: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor. - [Read A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. - [Read A Novel Fluorescent pH Probe for Expression in Plants]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO2 control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. - [Read A Sealed Preparation for Long-Term Observations of Cultured Cells]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Protocol on the details required of support slide construction for a sealed preparation for long-term observations of cultured cells. - [Read A Sealed Preparation for Long-Term Observations of Cultured Cells: Support Slide Construction]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

A simplified system for rapid generation of recombinant adenoviruses. Includes: Generation of Recombinants in Bacterial Cells; Viral Production in 293 or 911 Cells; Preparation of High Titer Viral Stocks. - [Read A Simplified System for Rapid Generation of Recombinant Adenoviruses]

Category: Nucleic Acid Purification Protocols

Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue]

Category: Plant Biology Protocols: Plant Genetics Protocols

For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable. Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used. Includes metaphase arresting reagents. - [Read Accumulation and Fixation of Plant Metaphase Chromosomes Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

Phalloidin binds specifically to F-actin, and fluorescent-tagged phalloidin stains the actin skeleton in cells in a manner that is very close to the staining pattern seen using anti-actin antibody. - [Read Actin Staining in Fixed Yeast Cells Protocol]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

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

There are many ways to adapt cell lines to serum-free media. Five methods are presented that are designed for adapting hybridomas to a protein-free medium. These protocols may require some modifications for your particular cell line and conditions. - [Read Adapting Cells to a Serum-Free Environment Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Protocol descibes the use of L929 mouse fibroblast cells cultured in vitro in an agarose overlay assay to assess the toxicity of test substances.  The assay may be useful in assessing the irritation potential of test substances (e.g. surfactant-based products) as an alternative to the Draize rabbit eye test. - [Read Agarose Overlay Assay Protocol]

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

Aggregation of ES cells and eight cell stage embryos. Bowtell Lab. - [Read Aggregation of ES cells and eight cell stage embryos]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Protocol for the aggregation of ES cells with mouse morula-stage embryos. Includes: Preparation of aggregation walls; ES cell preparation; Embryo preparation and aggregation. - [Read Aggregation of ES Cells with Mouse Morula- Stage Embryos Protocol]

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

Aggregation of ES Cells with Mouse Morula-state Embryos. Oxford Practical Series Approach. - [Read Aggregation of ES Cells with Mouse Morula-state Embryos]

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

Efficient derivation of mESCs. Bryja et al., 2005 Stem Cells Express. - [Read An efficient method for the derivation of mouse embryonic stem cells]

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

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

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: 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 fixed apoptotic cells. Flow cytometry reveals apoptotic nuclei in the subdiploid region of the cell cycle histogram... - [Read Analysis of DNA Fragmentation Using Propidium Iodide (PI) Staining After Ethanol Fixation Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

Category: Cell Organelle Protocols: Mitochondria Protocols

The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. - [Read Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1]

Category: RNAi Technology Protocols

Protocol describes a procedure to anneal sense and antisense siRNA strands to form a duplex.  The duplexes can then be transfected into cultured cells. - [Read Annealing siRNAs to Produce siRNA Duplexes Protocol]

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

Annexin V, belonging to a recently discovered family of proteins, the annexins, with anticoagulant properties has proven to be a useful tool in detecting apoptotic cells since it preferentially binds to negatively charged phospholipids like PS in the presence of Ca2+ and shows minimal binding to phosphatidylcholine and sphingomyeline. Changes in PS asymmetry, which is analyzed by measuring Annexin V binding to the cell membrane, were detected before morphological changes associated with... - [Read Annexin V Protocol]