Click on above link for entire protocol.
1. Collect blood (75 microliters) into 1ml PBS containing 5 microM EDTA (10 microliters of 0.5 M stock) and mix immediately to prevent clotting. Keep tubes on ice.

2. Remove RBCs from samples. This can be accomplished by several means. At The Jackson Laboratory, RBCs are lysed using either Gey’s solution or a buffered ammonium chloride (ACK) solution. (Alternatively, Becton-Dickinson sells a product called “FACS lysis buffer” that is used after the staining protocol to lyse RBCs and fix the cells.)

Susan Forsburg’s method for Schizosaccharomyces pombe

Reagents and Protocol
1. Spin down 107 cells from an exponentially growing culture - 2000 rpm for 5 mins. Pour off supernatant.

For the rest of the protocol please click on the above link.

Intracellular Immunofluorescent Staining for Flow Cytometric Analysis (FACS Analysis)

A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at the single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens as in the surface antigen protocol, then fixed with paraformaldehyde to stabilize the cell membrane and permeabilized with the detergent saponin to allow anti-cytokine antibodies to stain intracellularly.

Advanced Methodologies in Flow Cytometry

Andrea Cossarizza, Editor

Introduction   A. Cossarizza
Workshop Sponsors
Methods in analysis of apoptosis and cell necrosis.  Z. Darzynkiewicz
Common methods for measuring apoptosis cell death.  I. Nicoletti, et al.
Multiparametric flow cytometry for cell proliferation studies.  G. Mazzini, et al.
Multiparametric flow cytometry for determining DNA content in whole cells.  M. Carbonari, et al.
Flow cytometric immunophenotyping of HIV infected subjects and quality control.  A. Kunkl
Phenotypic and functional characterization of tumor infiltration lymphocytes: detection of an oligoclonal population with peculiar features.  S. Bruno, et al.
A multiparametric approach to immunophenotyping.  C. Ortolani
Triggering of Ca2+ flux by receptor crosslinking in hematopoietic cell.  R. De Maria, et al.
Three tips in methodologies.  G. Basso
NK function in flow cytometry.  M. Vitale
Measure of mitochondria membrane potential with the fluorescent probe JC-1.  A. Cossarizza

Flow Cytometry Analysis

by Qing Ma, 6/22/2000

Purpose

Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology can provide rapid, quantitative, multiparameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission. Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules.

Sugarbaker EV, Thornthwaite JT, Temple WT, Ketcham AS. Int Adv Surg Oncol. 1979;2:125-53.

The cell populations derived from normal tissues and solid tumors comprised many different cell types. Within each cell type there is a distribution of cells in different phases of the cell cycle and/or metabolic states (ie, differing rates of protein, RNA, and other macromolecular syntheses). Flow cytometry and companion instrumentation now promise to aid in rapid quantitative analyses of heterogeneous cell populations, thus finding broad applicability in many areas of cancer research and treatment. Since it is projected that this analytical technique will greatly expend our knowledge in tumor biology, it seems appropriate to review the basis principles of the methodology and to demonstrate recent applications in several areas of current research. After reviewing basis principles, a detailed description of one specific flow cytometer, the PHYWE-ICP-22, with its computer interface as developed in this laboratory is described. Subsequently, applications of this methodology to analyses of tumor cell kinetics, assays of blastogenesis, and studies of human colon cancer are presented as specific, current applications of flow cytometry. It is anticipated that this overview of flow cytometry along with some current applications will provide a background understanding for the inevitable rapid future developments in this area of research.