JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.
1Department of Physiology, Medical College of Wisconsin
Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.
Published June 27, 2015. Keywords: Molecular Biology, glomeruli, podocytes, calcium imaging, ion channels, confocal microscopy, intracellular calcium.
1Biological Effects Department, Centre for Radiation, Chemicals and Environmental Hazards, Public Health England
We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.
Published June 18, 2015. Keywords: Cellular Biology, Atherosclerosis, Endothelial cells, Monocytes, Adhesion, Inflammation, Adhesion assay
1Immunity and Infection Research Centre, Vancouver Costal Health Research Institute, 2Department of Surgery, University of British Columbia, 3Department of Biochemistry and Molecular Biology, University of British Columbia
Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.
Published June 18, 2015. Keywords: Cellular Biology, Transfection, RAW264.7, macrophages, luciferase, lipopolysaacharide, Interleukin-10, lipid-based transfection, polyamine-based transfection
1Department of Natural Sciences, Marymount Manhattan College
A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.
Published June 18, 2015. Keywords: Cancer Biology, Cell cycle, cell signaling, cancer, laboratory module, mouse mammary tumor cells, MMT cells, undergraduate, open-ended inquiry, breast cancer, cell-counting, cell viability, microscopy, science education, cell culture, teaching lab
1Cellular and Molecular Medicine Program, Boston Children’s Hospital and Harvard Medical School, 2Department of Medicine, University of Fribourg, 3Centre Médical Universitaire, University of Geneva
We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.
Published June 10, 2015. Keywords: Biochemistry, Granzyme, immune serine protease, cell-mediated cytotoxicity, recombinant protein production, mammalian expression system, protein purification
1Department of Biological, Geological, and Environmental Sciences, Cleveland State University
Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.
Published June 10, 2015. Keywords: Biochemistry, Cell free in vitro transcription-translation, HeLa cell free expression, rhoptry proteins, mammalian cell free expression system, Plasmodium falciparum, Pro Bond affinity purification
1Departments of Physiology, Medical College of Wisconsin, 2Human and Molecular Genetics Center, Medical College of Wisconsin, 3Cardiovascular Center, Medical College of Wisconsin, 4Blood Research Institute of Wisconsin
Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.
Published June 10, 2015. Keywords: Cellular Biology, Two-photon microscopy, fluorescence, vasculature, intracellular calcium, Fluo-4 AM, nitric oxide, DAF-FM, aorta, rat
1Center for Advanced Preclinical Research (CAPR), Frederick National Laboratory for Cancer Research, 2Leidos Biomedical Research, Inc., 3Department of Oncology, Georgetown University
The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.
Published May 31, 2015. Keywords: Structural Biology, Tissue Microarrays, Multi-Tissue Paraffin Blocks, Tissue Orientation, Histology, Immunohistochemistry
1Department of Biochemistry and Molecular Pharmacology, Institute for Systems Genetics, 2Roche Life Science, USA
Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.
Published May 25, 2015. Keywords: Molecular Biology, Sc2.0, synthetic biology, Saccharomyces cerevisiae, genotyping, PCRTag, Cobra, Echo, LightCycler 1536, real time PCR
1School of Life Sciences, Arizona State University, 2Biodesign Institute, Arizona State University
Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.…
Published May 25, 2015. Keywords: Molecular Biology, microRNA, luciferase assay, 3' untranslated region, high-throughput, transfection, post-transcriptional gene regulation, cancer