A contribution towards making this increasingly valuable technology accessible to researchers, including the students, post-doctoral scholars, and technicians gathering the knowledge inherent in this integration between analysis and physical isolation/purification methodologies. A step-by-step approach to the methodology for measuring various attributes demonstrated in the particular cells of interest is provided, as is a myriad of resources to fuel the curiosity and answer questions of both new and adept users. This book stems from the editors'experiences managing flow cytometry/cell sorting core facilities for the emerging researchers, in particular in developmental, cellular, and molecular biology.

Inhalt

Section 1.- A Palette of Living Colors.- The Colorful History of Flow Cytometry.- Section 2.- Getting Started.- 1 How the FACSCalibur and FACS Vantage Work and Why It Matters.- 2 How Flow Cytometers Work - and Don't Work.- 3 Flow Cytometry Standard (FCS) Data File Format.- 4 Options for Data Display.- 5 Guidelines to Improve Flow Cytometry Data Display and Interpretation.- 6 Data Management and Storage.- 7 Coloring up: A Guide to Spectral Compensation.- 8 Quality Control Guidelines for Research Flow Cytometry.- Section 3.- Sample Preparation and Cell Surface Staining.- 1 Cell Preparation and Enrichment for FCM Analysis and Cell Sorting.- 2 The Basics of Staining for Cell Surface Proteins.- 3 Phenotypic Analysis Using Five-Colour Immunofluorescence and Flow Cytometry.- 4 Detection of Cell Surface Antigens Using Antibody- Conjugated Fluorospheres and Flow Cytometry.- 5 Gel Microdrop Encapsulation for the Frugal Investigator.- Section 4.- Reporter Genes and Cell Trackers.- 1 Flow Cytometric Analysis of GFP Expression in Mammalian Cells.- 2 FACS-Gal: Flow Cytometric Assay of ß-galactosidase in Viable Cells.- 3 Utilization of a Bicistronic Expression Vector for Analysis of Cell Cycle Kinetics of Cytotoxic and Growth-arrest Genes.- 4 NGFR as a Selectable Cell Surface Reporter.- 5 Assessment of Cell Migration in vivo Using the Fluorescent Tracking Dye PKH26 and Flow Cytometry.- 6 In vivo Biotinylation for Cell Tracking and Survival/Lifespan Measurements.- 7 Enrichment and Selection for Reporter Gene Expression with MACSelect.- 8 Use of PKH Membrane Intercalating Dyes to Monitor Cell Trafficking and Function.- Section 5.- Flow Cytometry of Nucleic Acids.- 1 RNA Content Determination Using Pyronin Y.- 2 DNA Content Determination.- 3 Dead Cell Discrimination.- 4 Detecting Apoptosis.- 5 Chromosome Preparation - Polyamine Method.- 6 'D-Flowering' - The Flow Cytometry of Plant DNA.- 7 Flow Cytometry Analysis of Marine Picoplankton.- 8 Yeast DNA Flow Cytometry.- Section 6.- Intracellular Physiological and Antibody Probes.- 1 Calcium Flux Protocol: INDO-1 Stained Thymocytes.- 2 Intracellular pH Measured by ADB.- 3 Measuring Intracellular pH Using SNARF-1.- 4 FACS Analysis of Ph and Apoptosis.- 5 Measurement of Ligand Acidification Kinetics for Adherent and Non-Adherent Cells.- 6 Intracellular Antigen Detection by Flow Cytometry.- 7 Flow Cytometric Measurement of Nuclear Matrix Proteins.- 8 CellProbe Flow Cytoenzymology.- 9 Intracellular Cytokine Detection.- Section 7.- Electronic Cell Sorting.- 1 Creating Standard and Reproducible Sorting Conditions.- 2 Sterilization for Sorting.- 3 High Speed Cell Sorting Using the Cytomation CICERO® and MoFlo® Systems.- 4 Purification of Mouse Fetal Liver Hematopoietic Stem Cells.- 5 Methods for Preparing Sorted Cells as Monolayer Specimens.- 6 Fluorescent in Situ Hybridization Analysis on Cells Sorted onto Slides.- Section 8.- Core Facilities.- 1 Core Facility Management.- 2 Funding for Core Facilities.- 3 Biosafety in the Flow Cytometry Laboratory.- Section 9.- Unlocking the Spectrum of Possibilities.- Appendices.