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流式细胞仪交流论坛：http:/ 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_C
3、Y5.5 480 695 PerCP 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氦氖激光器荧光染料谱
4、染料名称 Excitation(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
5、551 Lucifer yellow 425 528 NBD 466 539 294 NBD-XR-Phycoerythrin (PE)480;565 578 240 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 con
6、jugateFluorX 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)650 660 104 k APC-Cy7 conjugates650;755 767 PharRedAlexa Fluo
7、r 系列荧光染料Alexa Fluor 350 343 442 410 Alexa Fluor 405 401 421 1028 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 546 561 572 1079 QY 0.79Alexa Fluor 555 553 568 1250 QY 0.1Alexa Fluo
8、r 568 579 603 792 QY 0.69Alexa Fluor 594 591 618 820 QY 0.66Alexa Fluor 610 610 629 1285 Alexa Fluor 633 632 648 1200 Alexa Fluor 647 652 668 1300 QY 0.33Alexa Fluor 660 663 691 1100 Alexa Fluor 680 680 702 1150 Alexa Fluor 700 696 719 1400 Alexa Fluor 750 752 776 1300 Alexa Fluor 790 782 804 1750 C
9、y 系列荧光染料Cy2 489 506 714 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 0.28Cy5.5 675 694 1128 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
10、 624 AT-selective SYTOX Blue 431 480 400 DNA Chromomycin A3 445 575 CG-selective Mithramycin 445 575 YOYO-1 491 509 1271 Ethidium Bromide 493 620 394 Acridine Orange 503 530/640 DNA/RNASYTOX Green 504 523 600 DNA TOTO-1, TO-PRO-1509 533 Vital stain, TOTO: Cyanine Dimer TO-PRO: Cyanine MonomerThiazol
11、e Orange 510 530 Propidium Iodide (PI)536 617 668.4 LDS 751 543;590 712;607 472 DNA (543ex/712em), RNA (590ex/607em) 7-AAD 546 647 7-aminoactinomycin D, CG-selectiveSYTOX Orange 547 570 500 DNA TOTO-3, TO-PRO-3642 661 DRAQ5 647 681,697 413 usable excitation down to 488细胞功能探针Indo-1 361/330 490/405 10
12、10 AM ester. Low/High Ca+,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 0.18 0.27 monomerEBFP2 38
13、3 448 20 monomerAzurite 383 447 15 monomerGFPuv 385 508 T-Sapphire 399 511 0.60 26 25 weak dimerCerulean 433 475 0.62 27 36 weak dimermCFP 433 475 0.40 13 64 monomerECFP 434 477 0.15 3 CyPet 435 477 0.51 18 59 weak dimerY66W 436 485 mKeima-Red 440 620 0.24 3 monomerTagCFP 458 480 29 dimerAmCyan1 458
14、 489 0.75 29 tetramermTFP1 462 492 54 dimerS65A 471 504 Midoriishi Cyan 472 495 0.9 25 dimerWild Type GFP 396,475 508 26k 0.77 S65C 479 507 TurboGFP 482 502 26 k 0.53 37 dimerTagGFP 482 505 34 monomerS65L 484 510 Emerald 487 509 0.68 39 0.69 weak dimer; (Invitrogen)S65T 488 511 EGFP 488 507 26k 0.60
15、 34 174 weak dimerAzami Green 492 505 0.74 41 monomerZsGreen1 493 505 105k 0.91 40 tetramerTagYFP 508 524 47 monomerEYFP 514 527 26k 0.61 51 60 weak dimerTopaz 514 527 57 monomerVenus 515 528 0.57 53 15 weak dimermCitrine 516 529 0.76 59 49 monomerYPet 517 530 0.77 80 49 weak dimerTurboYFP 525 538 2
16、6 k 0.53 1.65 dimerZsYellow1 529 539 0.65 13 tetramerKusabira Orange 548 559 0.60 31 monomermOrange 548 562 0.69 49 9 monomermKO 548 559 0.60 31 122 monomerTurboRFP 553 574 26 k 0.67 62 dimertdTomato 554 581 0.69 95 98tandem dimerTagRFP 555 584 50 monomerDsRed monomer 556 586 28k 0.1 3.5 16 monomerD
17、sRed2 (“RFP“) 563 582 110k 0.55 24 mStrawberry 574 596 0.29 26 15 monomerTurboFP602 574 602 26 k 0.35 26 dimerAsRed2 576 592 110k 0.21 13 tetramermRFP1 584 607 30k 0.25 monomerJ-Red 584 610 0.20 8.8 13 dimermCherry 587 610 0.22 16 96 monomerHcRed1 588 618 52k 0.03 0.6 dimerKatusha 588 635 23 dimermKate (TagFP635) 588 635 15 monomerTurboFP635 588 635 26 k 0.34 22 dimermPlum 590 649 0.10 4.1 53 mRaspberry 598 625 0.15 13 monomer; faster photobleach than mPlum其他探针Monochlorobimane 380 461 226 Glutathione probeCalcein 496 517 623 pH 5
