流式分析技术staining-cell-surface-antigens-for-flow-cytometry.pdfVIP

Flow Cytometry – BestProtocols® Page 1 of 4 Staining Cell Surface Antigens for Flow Cytometry Research Use Only  Protocol A: Cell Suspensions  Protocol B: Human Lysed Whole Blood Introduction Flow cytometry is a useful tool for simultaneously measuring multiple physical properties of individual particles (such as cells). Cells pass single-file through a laser beam. As each cell passes through the laser beam, the cytometer records how the cell or particle scatters incident laser light and emits fluorescence. Using this flow cytometric analysis protocol, one can perform a simultaneous analysis of surface molecules at the single-cell level. General Notes 1. For optimal performance of fluorochrome conjugated antibodies, store vials at 4°C in the dark. Do not freeze. 2. Prior to use, quick spin the antibody vial to recover the maximum volume. We do not recommend vortexing the antibody vial. 3. Except where noted in the protocol, all staining should be done on ice or at 4°C with minimal exposure to light. 4. Modifications relevant to staining with eFluor® nanocrystal (NC) reagents are noted in the general protocol by bold print. Useful websites Mario Roederers Home Page (/compensation/index.html) Mario Roederer is a key opinion leader in the field of flow cytometry. Purdue University Cytometry Laboratories (/index.htm) Flow Cytometry based public forum maintained by the Purdue University. Protocol A: Cell suspensions Materials  12x75 mm round bottom test tubes or 96-well round bottom microtiter plates  Primary antibodies (directly conjugated or purified)  Secondary reagents, if necessary (for indirect staining)  Affinity