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 Cytometry
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.
Excellent flow cytometry analysis protocol from the Springer Lab. Please follow the link for the entire protocol.
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.