流式細(xì)胞(FACS)多色組合原則和工具及新染料介紹ppt課件

1、多色組合原那么和工具新染料引見多色組合原那么按照機(jī)器設(shè)置染料的亮度與抗原表達(dá)相匹配同種細(xì)胞的Marker染料重疊最小化盡量防止結(jié)合運(yùn)用帶來的假陽性假設(shè)能夠，用紅激光做自發(fā)熒光高的樣本。染料選擇-按照機(jī)器設(shè)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表達(dá)強(qiáng)弱- Antigen Density染料選擇染料選擇熒

3、光染料的強(qiáng)度CD5CD8Example高表達(dá)的抗體可用不太亮的染料，低/弱表達(dá)的Marker用亮的染料Example: CD8 “bright Pacific BlueCD7 “l(fā)ess 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熒光染料光譜重疊最小化 spectra viewer光譜重疊會(huì)導(dǎo)致數(shù)據(jù)喪失CD45-FITCDim CD4-PECD45 FITC 漏到 PE， CD4 PE 弱信號(hào)分不開CD45- PerCPDim CD4-PECD45 PerCP 不漏到 PE ，弱 CD4 可以分開UncompensatedCompen

6、sateddata spread due to spillover采用多激光激發(fā)減少重疊同一細(xì)胞的抗原, 用不同激光激發(fā)減少重疊Example: CD3 “bright APC-Cy7CD7 “l(fā)ess bright PEBoth antigens expressed on same cell, low spillover of CD3 into CD7 and vice versa.CD3 = 124K molecules/cellCD7 = 20K molecules/cell雙激發(fā)熒光染料熒光染料可被不止1 laser激發(fā)，導(dǎo)致光譜重疊干擾，影響結(jié)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.熒光染料選擇防止染料和其衍生物 tandem-dye在同一細(xì)胞上標(biāo)志，或者選用更穩(wěn)定的tandem-dyeBD HorizonTMV500CD45BDTM APC-cy7CD14FITCCD8PerCP

8、-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigenBD HorizonTM V500CD45BDTM APC-cy7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigen不同細(xì)胞不同細(xì)胞由于 tandem-dye 降解會(huì)產(chǎn)生加陽性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補(bǔ)償: Tandems0 hours2 hours22.5 hoursPECD8CD3PE-Cy5PE-Cy7樣本曝光時(shí)間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自發(fā)熒光多的選擇用紅激光 染料選擇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 選擇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背景處置The Immunoglobulin背景處置-BlockingFcBlock Mouse FcBlock, purified CD16/32 cat # 553141/553142Reduces Ba

14、ckground StainingNo pre-inc. FcBlock Pre-inc. FcBlock多色運(yùn)用工具多色運(yùn)用工具28129230331323334353637383940414243PE-CF594激發(fā)Laser：488nm 或561nm發(fā)射波： 612 nml 更亮l 更穩(wěn)定l 溢漏更少FITCPEPE-CF594PerCPPE-CY744更亮的PE-CF59445更穩(wěn)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用規(guī)范的Horizon V450 filter: 450/50 nm48Brilliant Violet 421 ：極亮49極亮：更好的細(xì)胞分群亮的染料使細(xì)胞分群更明顯陽性率添加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穩(wěn)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 特點(diǎn)總結(jié) 極亮溢漏更少穩(wěn)定54運(yùn)用：MulticolorBV421是多色組合的理想選擇尤其是弱表達(dá)/低表達(dá)Panels FACSVerse很亮的很亮的10色組合色組合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損傷和細(xì)胞增殖Apoptosis- cleaved PARP (PE)DNA Damage- H2AX (Alexa Fluor 647)Cell Proliferati