Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80°C while attached to the substratum of 25cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.
Cryopreservation; Culture Media, Serum-Free; Cells, Cultured
Pancreatic β-cell apoptosis is known to participate in the β-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).
Apoptosis; Diabetes Mellitus; Streptozotocin; Cytokines
Several pathological and disease conditions can alter the mechanical properties of the extracellular matrix (ECM). Conversely, some diseases may arise from changes in the density or rigidity of the ECM. This necessitates the use and development of in vitro models to understand how both biophysical and biochemical signals regulate complex cellular behaviors. T47D breast epithelial cells will differentiate into duct-like tubules when cultured in a floating three-dimensional (3D) collagen gel, but not a 3D collagen gel that is left attached to the culture dish. This paper details several protocols we have developed for analyzing breast cell biology in 3D matrices, including culturing cells in 3D collagen gels, immunostaining cellular structures, and performing biochemical procedures directly from cells embedded in collagen gels.
Cell Culture Techniques; Signal Transduction; Mechanotransduction, Cellular; Epithelial Cells
We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.
Spectrum Analysis, Mass; Rhizobium; Electrophoresis, Gel, Two-Dimensional
Most behavioral experiments within circadian research are based on the analysis of locomotor activity. This paper introduces scientists to chronobiology by explaining the basic terminology used within the field. Furthermore, it aims to assist in designing, carrying out, and evaluating wheel-running experiments with rodents, particularly mice. Since light is an easily applicable stimulus that provokes strong effects on clock phase, the paper focuses on the application of different lighting conditions.
Photoperiod; Chronobiology; Circadian Rhythm; Mice
We have developed a simple and effective method (Lig-PCR) for monitoring ligation reactions using PCR and primers that are common to many cloning vectors. Ligation mixtures can directly be used as templates and the results can be analyzed by conventional gel electrophoresis. The PCR products are representative of the recombinant molecules created during ligation and the corresponding transformants. Orientation of inserts can also be determined using an internal primer. The usefulness of this method has been demonstrated using ligation mixtures of two cDNA’s derived from the salivary glands of Aedes aegypti mosquitoes. The method described here is sensitive and easy to perform compared to currently available methods.
Ligation; Nucleic Acids; Polymerase Chain Reaction
We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead.
Immunohistochemistry; Sheep; Gene Expression; Polymerase Chain Reaction
The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes.
Chromatin; Cross-linking Reagents; Formaldehyde; Histones; Immunoprecipitation
Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.
Apoptosis; Macrophage Colony-Stimulating Factor; Osteoclasts
Angiogenesis is a complex process involving an ECM and vascular endothelial cells (EC), and is regulated by various angiogenic factors including VEGF. The ability to form a capillary/tube-like network is a specialized function of EC. Therefore, in vitro angiogenesis was assessed by a capillary/tube-like network formation assay. There are three angiogenic parameters: capillary length, number of capillaries, and relative capillary area per field. We evaluated capillary length per field in the assay. VEGF promoted capillary/tube-like network formation of EC in a type I collagen gel matrix in vitro. Moreover, we demonstrated the involvement of ILK in a VEGF signaling pathway mediating capillary/tube-like network formation of EC using dominant-negative, kinase deficient ILK. This is a straightforward assay to monitor responses of human vascular endothelial cells.
Endothelial Cells; Vascular Endothelial Growth Factor A; Angiogenesis Inducing Agents
We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ~150 μm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix.
Microelectrode; Ion-Selective Electrodes
To date, breast cancer (BC) research is mainly studied with cell lines. These cells were passaged multiple times, acquiring phenotypes, additional mutations and epigenetic changes. These changes make the passaged cell lines different from the original malignancy. Thus cell lines, although useful as models could be improved with additional studies with primary BC. It is difficult to obtain malignant cells from breast tissues without contamination from surrounding healthy cells. Selection and expansion of malignant cells from surgical tissues have proved to be daunting tasks. This study describes a reliable and reproducible method for isolating and expanding malignant cells from surgical breast tissues. The method uses co-cultures with BM stroma to select for the cancer cells while the healthy cells undergo rapid cell death. Studies are described to show the cloning efficiencies and sensitivity of the method using surgical samples of varying sizes, different stages of BC, and samples from needle biopsies.
Bone marrow; Cultured, cells; Breast neoplasms
RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin β4 (Tβ4); determined their effectiveness at silencing Tβ4 and have both excluded off-target silencing of the Tβ4 homologue Thymosin β10 (Tβ10) and demonstrated partial knockdown of Tβ10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.
Thymosin beta(4); Thymosin beta(10)
To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.
Sortilin; Sphingolipid Activator Proteins