流式细胞术分析及应用.pptVIP

谢 谢！ * * The sample stream is injected into the center of the sheath steam in the optical cuvette and the particles are carried upward in a single file past the laser beam. This results in what is known as a laminar flow, where the sample stream is centered within the sheath, and it does not mix with the surrounding sheath fluid. The appropriate pressurization of the sheath and the sample are important for maintaining the laminar flow. The sample pressure is always slightly greater than the sheath pressure. The difference in pressure between the sample stream and sheath fluid stream can be used to vary the diameter of the sample stream. Increasing the sample pressure increases the core diameter and therefore the flow rate. A higher flow rate is generally used for measurements such as immunophenotyping. The data is less resolved but is acquired more quickly. A lower flow rate is generally used in applications where greater resolution is critical. * 1、荧光信号的面积：对荧光光通量进行积分 2 、DNA指数(DI)：相对DNA含量= 样品G0/G1细胞DNA含量平均数 标准二倍体DNA量的平均数 3、异倍体：非二倍体，包括近二倍体、四倍体、多倍体、非整倍体 凋亡细胞的特点 在形态上，早期细胞核固缩，染色体边集在核膜内侧显新月体形、核碎裂；细胞浆和细胞器密度增高、细胞体积变小；细胞膜皱折卷曲，但早期细胞膜的完整性未受到破坏 凋亡细胞的FSC降低，SSC增加。 细胞坏死时，细胞肿胀， FSC增大，SSC也增大 * Annexin V Assay * * Annexin V/PI双染 活细胞为（Annexin V-/PI-） 坏死细胞为（Annexin V+/PI+） 凋亡细胞（Annexin V+/PI-） Annexin V Assay----能够区分死亡与凋亡 * * 5、细胞因子测定 + + Antigen (cytokines) Fluorescence Detector antibody Capture beads Capture antibody Beads * Beads Provide a Flexible Platform Multiple sizes Beads provide an expandable assay platform for use with a flow cytometer Different intensities Different colors with different intensities * Multiplexed Beads Shades of a color Antibody coupled beads, emitting at distinct FL3 intensities Various analytes Antibody coupled PE label, emitting at FL2 intensity proportional to analyte conc. * Example 6-bead assay IL-8 IL-1b IL-6 IL-10 TNF IL-12 Capture Beads Lysate or Supernatant Sample Detector Antibodies Wash Analyze on flow cytometer * CBA Standar