流式细胞(FACS)多色组合原则和工具及新染料介绍ppt课件

1、多色组合原那么和工具新染料引见多色组合原那么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防止结合运用带来的假阳性假设能够，用红激光做自发荧光高的样本。染料选择-按照机器设置6-color8-colorAdditionalFITC or Alexa 488FITC or Alexa 488FITC or Alexa 488PEPEPEPE-CF594 or PE-Texas Red or PE-Alexa 610/594PE-CF594 or PE-Texas Red or PE-Alexa 610/594PerCP/PE-Cy5/PerCP-Cy5.5PerC

2、P/PE-Cy5/PerCP-Cy5.5PerCP/PerCP-Cy5.5/PE-Cy5/PE-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC or Alexa 647APC or Alexa 647APC or Alexa 647APC-Cy5.5/Alexa 680 or Alexa 700APC-Cy5.5/Alexa 680 or Alexa 700APC-H7/APC-Cy7APC-H7/APC-Cy7APC-H7/APC-Cy7BV 421Horizon V450/V500Pacific Orange, Q-dots表达强弱- Antigen Density染料选择染料选择荧

3、光染料的强度CD5CD8Example高表达的抗体可用不太亮的染料，低/弱表达的Marker用亮的染料Example: CD8 “bright Pacific BlueCD7 “less bright PECD8 = 90K molecules/cellCD7 = 20K molecules/cellEight color antibody panels proposed by the Human Immunophenotyping Consortium Nat Rev Immunol, 20217The importance of antibody choice The staining p

4、atterns of two commercially available clones of human CD38 specific antibody are very different, despite the fact that both antibodies were conjugated to allophycocyanin (APC) by the same vendor, and were used to stain peripheral blood mononuclear cells (PBMCs) from the same healthy subject under id

5、entical conditions. V450, violet 450. Data courtesy of Angelique Biancotto, National Heart, Lung and Blood Institute, USA. Nat Rev Immunol, 20218荧光染料光谱重叠最小化 spectra viewer光谱重叠会导致数据丧失CD45-FITCDim CD4-PECD45 FITC 漏到 PE， CD4 PE 弱信号分不开CD45- PerCPDim CD4-PECD45 PerCP 不漏到 PE ，弱 CD4 可以分开UncompensatedCompen

6、sateddata spread due to spillover采用多激光激发减少重叠同一细胞的抗原, 用不同激光激发减少重叠Example: CD3 “bright APC-Cy7CD7 “less bright PEBoth antigens expressed on same cell, low spillover of CD3 into CD7 and vice versa.CD3 = 124K molecules/cellCD7 = 20K molecules/cell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 Violet40

7、5nm) 和Blue488nm) (FITC detector).PE-Cy5 可漏到 APC detector.Without CD45 AmCyan:With CD45 AmCyan:CD19 FITCOnly an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.荧光染料选择防止染料和其衍生物 tandem-dye在同一细胞上标志，或者选用更稳定的tandem-dyeBD HorizonTMV500CD45BDTM APC-cy7CD14FITCCD8PerCP

8、-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigenBD HorizonTM V500CD45BDTM APC-cy7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigen不同细胞不同细胞由于 tandem-dye 降解会产生加阳性A.False positives inAPC channel reducedin absence of APC-Cy7False positives

9、in PE channelremainCD8 APC-Cy7+ cells CD4 PE-Cy7+ cellsB.With CD8 APC-Cy7 and CD4 PE-Cy7Without CD8 APC-Cy7补偿: Tandems0 hours2 hours22.5 hoursPECD8CD3PE-Cy5PE-Cy7样本曝光时间PerCPNew Tandems Are More StableAPC-H7 to replace APC-Cy7:Comparison of Sample Stability(in BD Stabilizing Fixative at RT)0501001502

10、002500124682448Hours of light exposure% SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy7自发荧光多的选择用红激光 染料选择BD HorizonTMV500CD45BDTM APC-H7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigenPerfect!Identification of immune cell subsets by eight colour antibody s

11、taining Nat Rev Immunol,202118Identification of immune cell subsets by eight colour antibody staining Nat Rev Immunol,202119Eight-color for hematopoietic stem cell MPPs: multipotent progenitor cellsCMPs: common myeloid progenitor cellsCLPs: common lymphoid progenitor cellsMEPs: megakaryocyte erythro

12、id progenitor cells GMPs: granulocyte macrophage progenitor cells20Eight-color for hematopoietic stem cell21Eight-color for hematopoietic stem cell22Six-color for hematopoietic stem cell23Buffer 选择Fixation bufferBD Cytofix/Cytoperm and BD Perm/Wash bufferBD Pharmingen FoxP3 buffer set (mouse or huma

13、n)BD Phosflow Perm Buffer IIBD Phosflow Perm Buffer IIIBD IntraSure kit Effect of BD Cytofix/Cytoperm Buffer on Mouse Foxp3 StainingMouse Foxp3 Alexa Fluor 647Foxp3 BufferCD4 PEBD Cytofix/Cytoperm背景处置The Immunoglobulin背景处置-BlockingFcBlock Mouse FcBlock, purified CD16/32 cat # 553141/553142Reduces Ba

14、ckground StainingNo pre-inc. FcBlock Pre-inc. FcBlock多色运用工具多色运用工具28129230331323334353637383940414243PE-CF594激发Laser：488nm 或561nm发射波： 612 nml 更亮l 更稳定l 溢漏更少FITCPEPE-CF594PerCPPE-CY744更亮的PE-CF59445更稳定的PE-CF594PE-CF594 reagents have consistent spillover values between lots and specificities, minimizing

15、the need for lot specific compensation controls46Brilliant Violet 421Ex Max: 407 nmEm Max: 421nm and 448 nm用规范的Horizon V450 filter: 450/50 nm48Brilliant Violet 421 ：极亮49极亮：更好的细胞分群亮的染料使细胞分群更明显阳性率添加PerCP-Cy5.5CD279PEBrilliant Violet42120%26%31%CD18444%79%50 Treg Panel 1 2CD127 A647CD25 BV421CD25 PECD1

16、27 BV421CD127 vs CD2551溢漏更少BV421V450V50052稳定 ：Stability in PFA fixativesSample PrepSample PrepTesting CriteriaStability ResultsFix cells & store in Stain Buffer 4C in the darkAbility to resolve positive & negative populations 96 hoursStain Index does not change more than 25% of Time 072 hour

17、sFix cells & store in Fixative 4C in the darkAbility to resolve positive & negative populations 96 hoursStain Index does not change more than 25% of Time 048 hours (1% PFA)24 hours (4% PFA)53BV421 特点总结 极亮溢漏更少稳定54运用：MulticolorBV421是多色组合的理想选择尤其是弱表达/低表达Panels FACSVerse很亮的很亮的10色组合色组合55APC-H7APC-

18、H7Comparison of Sample Stability(in BD Stabilizing Fixative at RT)0501001502002500124682448Hours of light exposure% SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy756BD Horizon V450， BD Horizon V500BD Horizon V450， BD Horizon V500V450- Pacific BlueV500- Pacific Orange AmCyan57the 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 Proliferati