流式常用荧光染料.doc

1、如何选择流式细胞仪测定常用的荧光染料流式细胞仪测定常用的荧光染料有很多种,我们如何来选择流式细胞仪测定的常用荧光染料呢？首先要考虑，他们分子结构不同,激发光谱和发射光谱也不同,选择荧光染料时，必须依据流式细胞仪所配备的激光光源的发射光波长做为重点考虑。发射光波长，如：氩离子气体激光管,它的发射光波 488nm；氦氖离子气体激光管发射光波长 633nm；488nm 激光光源常用的荧光染料有 FITC（异硫氰酸荧光素）；PE（藻红蛋白）PI（碘化丙啶）CY5（化青素）preCP(叶绿素蛋白)ECD(藻红蛋白-得克萨斯红)等。激发光和发射光波长，如下：激发光波长（nm） 发射光峰值（nm）FITC

2、488 525（绿）PE 488 575（橙红）PI 488 630（橙红）ECD 488 610（红）CY5 488 675（深红）PreCP 488 675（深红）流式细胞仪中常用的荧光染料光学特征以及需要的激光器名 称 Ex 波长(nm) Em 波长(nm) 备 注 激光器Hoechst 33342 355 465 Hoechst 33258 365 465 DAPI 372 456 DNA 染色核酸标记Indo-1 350 405 细胞内钙离子标记351nm紫外激光器FITC 490 520 R_PE 480 578 PE_CY5 480 670 PE_CY5.5 480 695 Pe

3、rCP 490 675 Alexa Fluor 488 494 517 PE_Texas Red 480 613 标记抗体Rhodamine 123 500 540 线粒体标记Fluo-3 506 526 细胞内钙离子标记DiOC6(3) 480 501 内质网标记YOYO-1 490 510 Propidium Iodide 530 615 Acridine Orange 490 640 DNA 染色核酸标记488nm氩离子激光器APC 650 660 Alexa Fluor 647 650 668 CY5 650 667 标记抗体633nm氦氖激光器流式细胞仪交流论坛：http:/ Exc

4、itation(nm) Emission(nm) 分子量 备注信息Reactive and conjugated probesHydroxycoumarin 325 386 331 Succinimidyl esterAminocoumarin 350 445 330 Succinimidyl esterMethoxycoumarin 360 410 317 Succinimidyl esterCascade Blue (375);401 423 596 HydrazidePacific Blue 403 455 406 MaleimidePacific Orange 403 551 Luci

5、fer yellow 425 528 NBD 466 539 294 NBD-XR-Phycoerythrin (PE) 480;565 578240 k PE-Cy5 conjugates480;565;650670 aka Cychrome, R670, Tri-Color, Quantum RedPE-Cy7 conjugates480;565;743767 Red 613 480;565 613 PE-Texas RedPerCP 490 675 Peridinin chlorphyll proteinTruRed 490,675 695 PerCP-Cy5.5 conjugateFl

6、uorX 494 520 587 GE HealthcareFluorescein 495 519 389 FITC; pH sensitiveBODIPY-FL 503 512 TRITC 547 572 444 TRITCX-Rhodamine 570 576 548 XRITCLissamine Rhodamine B570 590 Texas Red 589 615 625 Sulfonyl chlorideAllophycocyanin (APC)650 660104 k APC-Cy7 conjugates 650;755 767 PharRedAlexa Fluor 系列荧光染料

7、Alexa Fluor 350 343 442 410 Alexa Fluor 405 401 4211028 Alexa Fluor 430 434 540 702 Alexa Fluor 488 499 519 643 QY 0.92Alexa Fluor 500 503 525 700 Alexa Fluor 514 517 542 714 Alexa Fluor 532 530 555 724 QY 0.61Alexa Fluor 546 561 5721079 QY 0.79Alexa Fluor 555 553 5681250 QY 0.1Alexa Fluor 568 579 6

8、03 792 QY 0.69Alexa Fluor 594 591 618 820 QY 0.66Alexa Fluor 610 610 6291285 Alexa Fluor 633 632 6481200 Alexa Fluor 647 652 6681300 QY 0.33Alexa Fluor 660 663 6911100 Alexa Fluor 680 680 7021150 Alexa Fluor 700 696 7191400 Alexa Fluor 750 752 7761300 Alexa Fluor 790 782 8041750 Cy 系列荧光染料Cy2 489 506

9、 714 QY 0.12Cy3 (512);550 570;(615) 767 QY 0.15Cy3B 558 572;(620) 658 QY 0.67Cy3.5 581 594;(640)1102 QY 0.15Cy5 (625);650 670 792 QY 0.28Cy5.5 675 6941128 QY 0.23Cy7 743 767 818 QY 0.28核酸相关探针染料Hoechst 33342 343 483 616 AT-selective DAPI 345 455 AT-selective Hoechst 33258 345 478 624 AT-selective SYT

10、OX Blue 431 480400 DNA Chromomycin A3 445 575 CG-selective Mithramycin 445 575 YOYO-1 491 5091271 Ethidium Bromide 493 620 394 Acridine Orange 503 530/640 DNA/RNASYTOX Green 504 523600 DNA TOTO-1, TO-PRO-1 509 533 Vital stain, TOTO: Cyanine Dimer TO-PRO: Cyanine MonomerThiazole Orange 510 530 Propid

11、ium Iodide (PI) 536 617668.4 LDS 751 543;590 712;607 472 DNA (543ex/712em), RNA (590ex/607em) 7-AAD 546 647 7-aminoactinomycin D, CG-selectiveSYTOX Orange 547 570500 DNA TOTO-3, TO-PRO-3 642 661 DRAQ5 647 681,697 413 usable excitation down to 488细胞功能探针Indo-1 361/330 490/4051010 AM ester. Low/High Ca

12、+,Fluo-3 506 526 855 AM ester. pH 6DCFH 505 535 529 27Dichorodihydrofluorescein, oxidized formDHR 505 534 346Dihydrorhodamine 123, oxidized form, light catalyzes oxidationSNARF 548/579 587/635 pH 6/9荧光蛋白QY BR PS Y66H 360 442 Y66F 360 508 EBFP 380 440 0.180.27monomerEBFP2 383 448 20 monomerAzurite 38

13、3 447 15 monomerGFPuv 385 508 T-Sapphire 399 511 0.6026 25 weak dimerCerulean 433 475 0.6227 36 weak dimermCFP 433 475 0.4013 64 monomerECFP 434 477 0.153 CyPet 435 477 0.5118 59 weak dimerY66W 436 485 mKeima-Red 440 620 0.243 monomerTagCFP 458 480 29 dimerAmCyan1 458 489 0.7529 tetramermTFP1 462 49

14、2 54 dimerS65A 471 504 Midoriishi Cyan 472 495 0.925 dimerWild Type GFP 396,475 508 26k0.77S65C 479 507 TurboGFP 482 502 26 k0.5337 dimerTagGFP 482 505 34 monomerS65L 484 510 Emerald 487 509 0.68390.69weak dimer; (Invitrogen)S65T 488 511 EGFP 488 507 26k 0.6034174 weak dimerAzami Green 492 505 0.744

15、1 monomerZsGreen1 493 505105k0.9140 tetramerTagYFP 508 524 47 monomerEYFP 514 527 26k0.6151 60 weak dimerTopaz 514 527 57 monomerVenus 515 528 0.5753 15 weak dimermCitrine 516 529 0.7659 49 monomerYPet 517 530 0.7780 49 weak dimerTurboYFP 525 538 26 k0.531.65dimerZsYellow1 529 539 0.6513 tetramerKus

16、abira Orange 548 559 0. 31 monomer60mOrange 548 562 0.6949 9 monomermKO 548 559 0.6031122 monomerTurboRFP 553 574 26 k0.6762 dimertdTomato 554 581 0.6995 98 tandem dimerTagRFP 555 584 50 monomerDsRed monomer 556 58628k0.13.516 monomerDsRed2 (“RFP“) 563 582110k0.5524 mStrawberry 574 596 0.2926 15 mon

17、omerTurboFP602 574 602 26 k0.3526 dimerAsRed2 576 592110k0.2113 tetramermRFP1 584 60730k0.25monomerJ-Red 584 610 0.208.813 dimermCherry 587 610 0.2216 96 monomerHcRed1 588 61852k0.030.6dimerKatusha 588 635 23 dimermKate (TagFP635) 588 635 15 monomerTurboFP635 588 635 26 k0.3422 dimermPlum 590 649 0. 4. 53 10 1mRaspberry 598 625 0.1513 monomer; faster photobleach than mPlum其他探针Monochlorobimane 380 461 226 Glutathione probeCalcein 496 517 623 pH 5