流式细胞内抗原和细胞因子检测stainingintracellularantigensforflow-cytometry.pdfVIP

Flow Cytometry – BestProtocols® Page 1 of 9 Staining Intracellular Antigens for Flow Cytometry Research Use Only  Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins  Protocol B: One-step protocol: intracellular (nuclear) proteins  Protocol C: Two-step protocol: Fixation/Methanol Introduction 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 by flow cytometry. Typically, cells are fixed with formaldehyde to stabilize the cell membrane, and then permeabilized with detergent or alcohol to allow antibodies against intracellular antigens access to stain intracellularly. When staining proteins inside the cell, it is important to consider their location as this may dictate the protocol and buffer system that will perform optimally. For example, nuclear proteins and many secreted proteins work well with the eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience Cat. No. 00-5523), while secreted proteins such as cytokines and chemokines work well with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience Cat. No. 88-8824). Lastly, there are several phosphorylated signaling proteins that may not work in the two previously-mentioned buffer systems but will work with the Fixation/Methanol Protocol. Information about performance and preferred buffers is noted on the specific product’s Technical Data Sheet. Please contact Technical Support (tech@) for more information. 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, quickly spin the antibody vial to recover the maximum volume. We do not