cellular Protocols

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: Protein Protocols: Protein Phosphorylation Protocols

A Strategy for the Rapid Identification of Phosphorylation Sites in the Phosphoproteome. MacDonald et al., Molecular & Cellular Proteomics. 2002. - [Read A Strategy for the Rapid Identification of]

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

Protocol for automated imaging-based high-throughput screening for small molecules that reverse cellular senescence. - [Read Automated Imaging-Based High-Throughput Screening: Small Molecules that Reverse Cellular Senescence]

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

Peroxisomes of higher eukaryotes, glycosomes of kinetoplastids, & glyoxysomes of plants are related microbody organelles that perform differing metabolic functions tailored to their cellular environments. The close evolutionary relationship of these organelles is most clearly evidenced by the conservation of proteins involved in matrix protein import and biogenesis. glycosome can be viewed as an offshoot of the peroxisomal lineage with additional metabolic functions, specifically glycolysi - [Read Biogenesis and Function of Peroxisomes and Glycosomes]

Category: Immunology: B Cell Protocols

B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. Provides methods for the latter (direct) approach--namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II-antigen expression. - [Read Early Events in B Lymphocyte Activation Protocol]

Category: Cell Biology and Cell Culture: Cell Electroporation Protocols

Electroporation of Cell Lines With DNA. Electroporation for the efficient transfection of mammalian cells with DNA. Chu et al. Cellular Immunology Oxford - [Read Electroporation of Cell Lines With DNA]

Category: Cell Biology and Cell Culture

Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients. - [Read Equilibrium Density Gradient Percoll Protocol]

Category: Histology Protocols: Fixation of Cells Tissues Protocol

Fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilises the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Great detailed guide with protocols. Anthony S-Y Leong. - [Read Fixation and Fixatives - A great guide]

Category: Cell Culture Protocols: Cell Fixing Protocols

Organic solvents such as alcohols and acetone remove lipids and dehydrate cells, precipitating the proteins on the cellular architecture. Be aware that different antigens may be affected differently by the various solvents. If no previous data are available for your antigen, start with the 50/50 mixture. For tissue culture dishes, concentrations of acetone higher than 50% will destroy the integrity of the plastic. - [Read Fixing Attached Cells in Organic Solvents Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti-sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent-labeled dUTP nucleotide base. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular.... - [Read Fluorescent In Situ Transcription in Cells and Tissues Protocol]

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

This method measures the leakage of DNA and lactate dehydrogenase from lymphocytes into the surrounding medium as an indicator of cytotoxicity.  This method also includes an assay of intracellular (mitochondrial) diaphorase as a measure of cellular activity (MTT assay). - [Read Human Lymphocyte Cytotoxicity Assay Protocol]

Category: Immunology: T Cell Protocols

Protocol for the Stimulation of human peripheral blood mononuclear cells with anti-human CD3 monoclonal antibody; MTT assay for detection of cellular proliferation. Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies. Performed titration studies with these antibodies and established the following protocol for stimulation of PBMC. - [Read Human T Cell Activation Protocol]

Category: PCR Protocols: In-situ PCR Protocols

Amplification and Detection in a Cellular Context. Methodology, in-situ detection methods, reaction Conditions, introduction. Ernest F. Retzel et al., University of Minnesota. - [Read In-Situ PCR Protocol]

Category: Avian Bird Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA).  The dsRNA is injected into the spinal cord of the embryo.  Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules.  It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi—cell competence changes with differentiation. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

Activation and inactivation of proteins using photoactivation of caged peptides or proteins offer insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group. - [Read Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide critical insight into the fundamental nature of cellular & tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein & synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology labs. Includes: Maintaining Live Cells on the Microscope Stage; Live-Cell Imaging Culture Chambers; Optical System and Detector Requirements etc. - [Read Introduction to Live-Cell Imaging Techniques]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the first step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LC-MS/MS.  This procedure is used to prepare protein extracts from WEHI-231 cells.  This preparation method provides total cellular protein samples that are free of contaminating nucleic acids. - [Read Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide a critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology laboratories. - [Read Maintaining Live Cells on the Microscope Stage]

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: Immunology: T Cell Protocols

Mouse Protocol: Stimulation of mouse peripheral T cells with plate-bound 145-2C11 monoclonal antibody; MTT assay for detection of cellular proliferation. - [Read MTT Assay for Detection of Cellular Proliferation]

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

The rate of cellular proliferation may be regarded as an overall indicator of the physiological status of the cell.  Therefore, the effect of various toxic substances on different cell functions will be reflected by changes in the proliferation rate. - [Read Ovary Cell Proliferation Test]

Category: RNAi Technology Protocols

Short interfering RNAs (siRNAs) can be used to prime RNA synthesis by the RNA-dependent RNA polymerase (RdRP).  SiRNAs can be used by RdRP as primers for specific cellular mRNAs, forming dsRNA products capable of inducing transitive RNAi. - [Read Protocol for siRNA-Primed RNA Synthesis Protocol]