BD-流式基础知识-qj.pptVIP

同型对照（Isotype control） 例：一个双色染色的实验 抗体A FITC，抗体B PE 对应的同型对照是IgG1 FITC,IgG1 PE 那么需要准备的是 阴性对照：细胞加上IgG1 FITC,IgG1 PE 单阳性对照： FITC: 细胞加上Ab A FITC PE: 细胞加上Ab B PE 同型抗体为没有特异性，与荧光抗体蛋白亚型一致的免疫球蛋白，通常是未进行免疫小鼠血清的纯化IgG1或IgG2. 同型对照就是使用同型抗体进行平行实验，反映待测样本对抗体非特异性结合水平。 数据分析 设门 设定阴性与阳性群体的界限 确定阳性与阴性细胞群体 统计阳性或阴性细胞群体的百分率，平均荧光值，绝对数或抗体结合数 如何设定阴性与阳性的界限 直方图 散点图 Thanks! * * * 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. * The optical system is made up of the light source (laser), the lenses and prisms that focus the laser light on to the sample, and the lenses that collect the scattered light and emitted fluorescence. The fluorescence signals need to be separated and sent to their respective detectors. Three types of detectors are used in the FACSArray system: PMTs (photo multiplier tube), photodiode (for FSC only) and APDs (Avalanche photodiode). FYI: In BD FACSArray bioanalyzers, an avalanche photodiode (APD) is used to detect the far red (eg, PE-CY7) and near infrared (eg, APC-CY7) signals because they are very sensitive to si