JoVE General
Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología-Universidad Nacional Autónoma de México, 2Math and Sciences Department, Edison State College
Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.
JoVE General
Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence
1Department of Medicine, Albert Einstein College of Medicine, 2Diabetes Research and Training Center, Albert Einstein College of Medicine, 3Department of Genetics, Albert Einstein College of Medicine
An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.
JoVE General
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Molecular, Cellular and Developmental Biology, University of Michigan
A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.
JoVE General
A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation
1Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Emory University School of Medicine and Children's Health Care of Atlanta, 2Division of Rheumatology, Department of Pediatrics, Emory University School of Medicine and Children's Health Care of Atlanta
We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.
JoVE General
Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique
1Meakins-Christie Laboratories, Department of Medicine, McGill University, 2SCIREQ Scientific Respiratory Equipment Inc.
The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).
JoVE General
Protein Transfection of Mouse Lung
Department of Medicine, St. Luke's Roosevelt Medical Center
Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.
JoVE General
Introduction to Solid Supported Membrane Based Electrophysiology
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, 2Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt
Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.
JoVE General
Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
1Biology Department, Western Washington University, 2Washington State University Northwestern Research and Extension Center, 3Department of Plant and Soil Science, Texas Tech University
Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.
JoVE General
A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice
1Division of Nephrology-Hypertension, Department of Medicine, University of California, San Diego, 2San Diego VA Healthcare System
Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.
JoVE General
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
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