流式细胞(FAC)多色组合原则和工具及新染料介绍.pptVIP

多色组合原则和工具新染料介绍 多色组合原则 按照机器设置 染料的亮度与抗原表达相匹配 同种细胞的Marker染料重叠最小化 尽量避免联合使用带来的假阳性 如果可能，用红激光做自发荧光高的样本。 染料选择-按照机器设置 表达强弱- Antigen Density 荧光染料的强度 Example 荧光染料光谱重叠最小化 spectra viewer 光谱重叠会导致数据丢失 采用多激光激发减少重叠 双激发荧光染料 荧光染料可被不止1 laser激发，导致光谱重叠干扰，影响结果. AmCyan 可被 Violet（405nm) 和Blue（488nm) (FITC detector). PE-Cy5 可漏到 APC detector. 荧光染料选择 避免染料和其衍生物（ tandem-dye）在同一细胞上标记，或者选用更稳定的tandem-dye 由于 tandem-dye 降解会产生加阳性 补偿: Tandems New Tandems Are More Stable 染料选择 Eight-color for hematopoietic stem cell Eight-color for hematopoietic stem cell Eight-color for hematopoietic stem cell Six-color for hematopoietic stem cell Buffer 选择 Fixation buffer BD Cytofix/Cytoperm? and BD? Perm/Wash buffer BD Pharmingen? FoxP3 buffer set (mouse or human) BD? Phosflow Perm Buffer II BD? Phosflow Perm Buffer III BD IntraSure? kit Effect of BD Cytofix/Cytoperm Buffer on Mouse Foxp3 Staining 背景处理 背景处理-Blocking 多色应用工具 PE-CF594 更亮的PE-CF594 更稳定的PE-CF594 PE-CF594 reagents have consistent spillover values between lots and specificities, minimizing the need for lot specific compensation controls Brilliant Violet? 421  Brilliant Violet? 421 Ex Max: 407 nm Em Max: 421nm and 448 nm 用标准的Horizon V450 filter: 450/50 nm Brilliant Violet? 421 ：极亮 极亮：更好的细胞分群 亮的染料使细胞分群更明显 阳性率增加 Treg Panel  1 2 溢漏更少 稳定 ：Stability in PFA fixatives BV421 特点总结 极亮 溢漏更少 稳定 应用：Multicolor BV421是多色组合的理想选择尤其是弱表达/低表达 Panels （FACSVerse） APC-H7 BD Horizon V450， BD Horizon V500 Apoptosis, DNA Damage and Cell Proliferation Kit the Apoptosis, DNA Damage and Cell Proliferation Kit 同一管分析凋亡、DNA损伤和细胞增殖 Apoptosis- cleaved PARP (PE) DNA Damage- H2AX (Alexa Fluor? 647) Cell Proliferation –BrdU (PerCP-Cy5.5) 省时、节约珍贵样本 Sample Data Viability：Fixable Viability Stain 450（Cat.no. 562247)  BD Horizon? Fixable Viability Stain 450 (FVS450) 区分死活细胞 With camptothecin treatment (bottom) cell proliferation goes down (BrdU+ cells), DNA damage goes up (H2AX+ cells), and apoptosis goes u