The Molecular Probes Handbook, 11th Edition 3, The Molecular Probes Handbook www. thermofisher.com/handbook Fluorescen

The Molecular Probes Handbook, 11th Edition 3, The Molecular Probes Handbook www. thermofisher.com/handbook www.thermofisher.com/antibodies Fluorescence SpectraViewer 14 www.thermofisher.com/spectraviewer BioProbes Journal and newsletter BioProbes Journal BioProbes Journal www.thermofisher. com/bioprobes NOVEMBER 215 BIOPROBES 72 MOLECULAR PROBES JOURNAL OF CELL BIOLOGY APPLICATIONS SPECIAL IMAGING ISSUE Visualize apoptosis in context with Click-iT Plus TUNEL assays ALSO FEATURING Intracellular detection of hypoxia Formation of uniform 3D cancer spheroids Protection from photobleaching for live cells www.thermofisher.com/flowresources Attune NxT Acoustic Focusing Cytometer www.thermofisher.com/attune Flow cytometry guide and protocols Fluorescence SpectraViewer www.thermofisher.com/probeswebinars Webinar!? www.thermofisher.com/imagingbasics facebook.com/thermofisherjapan DailyCalcs science calculator @ThermoFisherJP www.thermofisher.com/apps www.thermofisher.com/probes

Invitrogen 4 www.thermofisher.com/flow-cytometry 12 12 15 18 19 2 2 2 Dynabeads 3 6 6 7 7 9 2 22 24 25 28

2 Invitrogen TM www.thermofisher.com/ flow-sample TransFix Invitrogen TM TransFix TM FIX2 FIX2 FIX1 TransFix Cal-Lyse Whole Blood Lysing Solution Invitrogen TM Cal-Lyse TM GAS1 GAS1S1 Cal-Lyse High-Yield Lyse Invitrogen TM High-Yield Lyse HYL25 FIX & PERM Invitrogen TM FIX & PERM TM Cell Permeabilization Kit GAS3 GAS4Medium A Medium B 2 1 GAS1S1 GAS2S1 A TNF-α Pacific Blue fluorescence B TNF-α Pacific Blue fluorescence 1 6 1 5 1 4 1 3-1 3-1 3 1 3 1 4 1 5 1 6 IFN γ PE-Cy 7 fluorescence 1 6 1 5 1 4 1 3-1 3-1 3 1 3 1 4 1 5 1 6 CD4 Fluorescein fluorescence 1 3-1 3-1 3 1 3 1 4 1 5 1 6 IFN γ PE-Cy 7 fluorescence 1 3-1 3-1 3 1 3 1 4 1 5 1 6 CD4 Fluorescein fluorescence 1. FIX & PERM Cell Permeabilization Kit C57BL/6 2 A PMA 5 FITC CD4 MCD41 FIX & PERM Cell Permeabilization Kit GAS3 GAS4 PE-Cy 7 IFN- A18713 Pacific Blue TNF- RM9128 AttuneAcoustic Focusing Cytometer / 488 nm 53/3 nm FITC 64 nm PE-Cy 7 Pacific Blue 45 nm 45/4 nm A IFN- TNF- A PMA B TNF- IFN- CD4 + T TNF-α Pacific Blue fluorescence IFN γ PE-Cy 7 fluorescence 1 6 1 5 1 4 1 6 1 5 1 4

3 Dynabeads Dynabeads TM 2 depletion 3 Dynabeads 4 Dynabeads T B NK 1 2 Dynabeads Dynal MPC 3. Dynabeads 3 mrna/dna : Dynabeads Dynabeads Dynabeads Dynabeads www.thermofisher.com/humancellisolation 25 5 75 1, 1,25 5 1, 1,5 2, 1 2 1 3 1 4 1 5 CD4 FITC 1 2 1 3 1 4 1 5 CD4 FITC 4. CD4 + T Dynabeads Untouched TM Human CD4 T Cells Kit 2. Dynabeads

Dynabeads 5 Dynabeads T B NK 4 : Dynabeads Dynabeads Dynabeads Dynabeads www.thermofisher.com/mousecellisolation www.thermofisher.com/cellisolation Dynabeads 計 数計 数 6 5 4 3 2 1 4 35 3 25 2 15 1 5 Dynabeads Dynabeads FlowComp FlowComp Mouse Mouse CD4 Kit CD4 Kit 1 2 1 3 1 4 1 5 CD4 + 97.2% 78.5% ヨウ化プロピジウム ヨウ化プロピジウム 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 CD4 + CD4 + 5. CD4 + T CD4 + T Dynabeads FlowComp Mouse CD4 Kit 78% 63% 97% 86% 1 5 1 4 1 3 1 2 1 5 1 4 1 3 1 2 1 2 1 3 1 4 1 5 CD4 + 86% 63%

5 2 ml FIX2 TransFix NA NA 2 ml FIX2 1 ml FIX1 25 ml GAS1 Cal-Lyse Whole Blood Lysing Solution NA NA 1 ml GAS1S1 High-Yield Lyse NA NA 5 ml HYL25 FIX & PERM Cell Permeabilization Kit NA NA 5 GAS3 2 GAS4 Medium A NA NA 1 ml GAS1S1 Medium B NA NA 1 ml GAS2S1 Dynabeads Dynabeads FlowComp Human CD4 Kit T 3 ml 11361D Dynabeads CD4 Positive Isolation Kit T 5 ml 11331D Dynabeads Untouched Human CD4 T Cells Kit T 1 11352D Dynabeads FlowComp Human CD8 Kit T 3 ml 11362D Dynabeads CD8 Positive Isolation Kit T 5 ml 11333D Dynabeads Untouched Human CD8 T Cells Kit T 1 11348D Dynabeads FlowComp Mouse CD4 Kit T 3 ml 11461D DETACHaBEAD Mouse CD4 Kit T 5 ml 1246D Dynabeads Untouched Mouse CD4 Cells Kit T 2 x 1 ml 11415D Dynabeads FlowComp Mouse CD8 Kit T 1 11462D Dynabeads Untouched Mouse CD8 Cells Kit T 2 x 1 ml 11417D N/A Dynabeads 15-1 2-7-14 TEL 3-3593-3211FAX 3-3593-3216 E-mail veritas@veritastk.co.jp URL http://www.veritastk.co.jp

www. thermofisher.com/flow-standards Flow Cytometry Sub-micron Particle Size Reference Kit Invitrogen TM Flow Cytometry Sub-micron Particle Size Reference Kit F13839 6 Alexa Fluor 488 FITC 55 nm 515 nm 6 AlignFlow Cytometry Alignment Beads Invitrogen TM AlignFlow TM Flow Cytometry Alignment Beads 6 AlignFlow AlignFlow 35 37 nm UV A1652 A1655 488 nm A165 A1653 633 nm A1651 A1654 3 2.5 µm 6. µm 2 Flow Cytometry Size Calibration Kit Invitrogen TM Flow Cytometry Size Calibration Kit F13838 6 FSC 7 粒子の数 1 2 1 3 1 4 1 2 1 3 1 4 1 2 1 3 1 4 緑色 橙色 赤色 蛍光強度 6. 488 nm 3 AlignFlow 3 : Carleton Stewart, Rosewell Park Cancer Institute 粒子の数 3 2 1 2 1. µm 2. µm 4. µm 4 6 FSC 6. µm 1 µm 15 µm 8 1 7. Flow Cytometry Size Calibration Kit Flow Cytometry Size Calibration Kit F13838 6 FSC FSC 488 nm Becton Dickinson FACScan

7 FSC 8 2 6 Cell Sorting Set-Up Beads Cell Sorting Set-Up Beads C1656 C1657 C1658 C1659 6 µm 1% 2 9 Alexa Fluor 488 R- R- PE Alexa Fluor 488 Alexa Fluor 488 R-PE 2 1 Invitrogen TM AbC TM Bead Kit Invitrogen TM ArC TM Amine Reactive Compensation Bead Kit Invitrogen TM LIVE/DEAD TM Fixable Dead Cell Stain Kit 2 AbC compensation bead kits 1 53/3 nm BP 蛍光シグナル 1 6 1 5 1 4 1 3 1 2 1 1 1 1 2.5 µm.1 µm.2 µm 1 3 1 4 1 5 側方散乱光 1 µm 1 6 2 µm Relative intensity (%) 75 5 25 3 4 5 6 7 8 9 波長 (nm) 9. Alexa Fluor 488 R-PE 8. Flow Cytometry Sub-micron Particle Size Reference Kit Flow Cytometry Sub-micron Particle Size Reference Kit F13839 6 5 5 Attune Acoustic Focusing Cytometer 488 nm 53/3 nm BP 5

1. AbC Total Antibody Compensation Bead Kit AbC Anti-Mouse Bead Kit AbC Anti-Rat/Hamster Bead Kit * A1513 A1497 * A1344 * A1389 ArC Amine Reactive Compensation Bead Kit LIVE/DEAD Fixable Dead Cell Stain A1346 * AbC 8 AbC Bead Kit (1) AbC Total Antibody Compensation Bead Kit A1497 1 (2) AbC Anti-Mouse Bead Kit A1344 11 (3) AbC Anti-Rat/Hamster Bead Kit A1389 3 2 AbC TM 2 11 2 AbC 6 µm ArC Amine Reactive Compensation Bead Kit LIVE/DEAD Fixable Dead Cell Stain kit ArC Amine Reactive Compensation Bead Kit A1346 LIVE/DEAD Fixable Dead Cell Stain Kit LIVE/DEAD Fixable Dead Cell Stain Kit 5 A B C D 9 マウス IgG1 8 マウス IgG2a マウス IgG2b 7 マウス IgG3 6 マウス IgGM 5 4 3 2 1 1, 9 8 7 6 5 4 3 2 1 ラット IgG1 ラット IgG2a ラット IgG2b ラット IgG2c ラット IgGM 7 6 5 4 3 2 1 アルメニアンハムスター IgG シリアンハムスター IgG 8 7 6 5 4 3 2 1 ウサギモノクローナル IgG ウサギポリクローナル IgG 1 1 1 2 1 3 1 4 1 5 1 6 PE fluorescence 1 1 1 2 1 3 1 4 1 5 1 6 PE fluorescence 1 1 1 2 1 3 1 4 1 5 1 6 PE fluorescence 1 1 1 2 1 3 1 4 1 5 1 6 PE fluorescence 1. AbC Total Antibody Compensation Bead Kit A BCD PE 488 nm 574/26 nm Attune Acoustic Focusing Cytometer

CD3-FITC fluorescence 9 ArC Amine Reactive Compensation Bead Kit ArC A ArC TM B 2 LIVE/DEAD Fixable Dead Cell Stains 2 12 ArC Amine Reactive Compensation Bead Kit AbC Anti-Mouse Bead Kit LIVE/DEAD Fixable Dead Cell Stain Invitrogen TM Bright TM Absolute ing Beads AccuCheck ing Beads 2 2 A 25 B 25 C 2 15 1 5-1 2 1 2 1 3 1 4 1 5 PE fluorescence 2 15 1 5-35 -1 2 1 2 1 3 1 4 1 5 FITC fluorescence 1 5 1 4 1 3 1 2-1 2-1 2 1 2 1 3 1 4 1 5 CD56-PE fluorescence 11. AbC Anti-Mouse Bead Kit A AbC PE CD56 MHCD564 B AbC FITC CD3 MHCD314 C 2 PE CD56 FITC CD3 AbC TM Anti-Mouse Bead Kit A1344 A B C 4 35 3 25 2 15 1 5-1 2 1 2 1 3 1 4 1 5 LIVE/DEAD Fixable Violet stain fluorescence 35 3 25 2 15 1 5-288 -1 2 1 2 1 3 1 4 1 5 LIVE/DEAD Fixable Green stain fluorescence 35 3 25 2 15 1 5-715 -1 2 1 2 1 3 1 4 1 5 LIVE/DEAD Fixable Red stain fluorescence 12. 3 LIVE/DEAD Fixable Dead Cell Stain Kit ArC Amine Reactive Compensation Bead Kit A LIVE/DEAD Fixable Violet Dye L34955 45 nm 45/5 nm B LIVE/DEAD Fixable Green Dye L2311 488 nm 525/5 nm C LIVE/DEAD Fixable Far Red Dye L112 633 nm 66/2 nm

2. nm nm Bright Absolute ing Beads 7 µm UV 635 385 to 8 C3695 AccuCheck ing Beads Bead A: 6.4 µm Bead B: 6.36 µm Bead A: 488 Bead B: 635 Bead A: 575 585 Bead B: 66 68 PCB1 1 Bright Absolute ing Beads / Invitrogen TM Bright TM Absolute ing Beads C3695 UV 635 nm 385 8 nm 1, Bright Absolute ing Beads / 7 µm Bright AccuCheck ing Beads 2 AccuCheck ing Beads PCB1 2 A B 2 AccuCheck ing Beads / 1 AccuCheck ing Beads 2 EthD-1 fluorescence 1 5 1 4 1 3 1 2 死細胞 カウントビーズ 生細胞 1 2 1 3 1 4 Calcein fluorescence 1 5 13. Bright Absolute ing Beads Jurkat LIVE/DEAD Viability/Cytotoxicity Kit L3224 Bright Absolute ing Beads C3695 488 nm 53/3 nm 1 EthD-1 61 nm

11 Ex/Em* AlignFlow Flow Cytometry Alignment Beads for UV Lasers, 2.5 µm AlignFlow Flow Cytometry Alignment Beads for UV Lasers, 6. µm AlignFlow Flow Cytometry Alignment Beads for Blue Lasers, 2.5 µm AlignFlow Flow Cytometry Alignment Beads for Blue Lasers, 6. µm AlignFlow Flow Cytometry Alignment Beads for Red Lasers, 2.5 µm AlignFlow Flow Cytometry Alignment Beads for Red Lasers, 6. µm Flow Cytometry Size Calibration Kit Flow Cytometry Sub-micron Particle Size Reference Kit UV 35 37/ 4 47 3 ml 488/515 66 3 ml 633/645 68 3 ml A1652 A1655 A165 A1653 A1651 A1654 NA NA 1 F13838 55/515 1 F13839 Cell Sorting Set-Up Beads for UV Lasers UV 35 375/46 3 ml C1656 Cell Sorting Set-Up Beads for Blue Lasers 488/515 3 ml C1658 Cell Sorting Set-Up Beads for Green-Yellow Lasers / 532, 561/575 3 ml C1659 Cell Sorting Set-Up Beads for Red Lasers 633/68 3 ml C1657 AbC Total Antibody Compensation Bead Kit NA NA 1 tests 25 tests A1497 A1513 AbC Anti-Mouse Bead Kit NA NA 1 A1344 AbC Anti-Rat/Hamster Bead Kit NA NA 1 A1389 ArC Amine Reactive Compensation Bead Kit NA NA 1 A1346 AccuCheck ing Beads / 488/575-585 A 1 ml PCB1 Bright Absolute ing Beads UV UV to 635/385 to 8 5 ml C3695 N/A * Ex Em nm

A Invitrogen TM Qdot TM RUO ASR IVD 2, RUO/ASR/IVD CD www.thermofisher.com/flow-searchantibodies B 12 1975 Alexa Fluor Qdot Invitrogen TM Zenon TM 3 Alexa Fluor Invitrogen TM Pacific Blue TM Pacific Green TM Pacific Orange TM PE PerCP APC 11 3 14 4 C 14. Attune NxT Acoustic Focusing Cytometer T 1 4 C57BL/6 FIX & PERM Cell Permeabilization Kit GAS3 GAS4 PE Foxp3 FSC/SSC A B22 B A B22 - CD45.2 + CD3 T A CD4 CD8 CD3 + T 2 B CD4 + T Foxp3 CD25 IL-2R T B CD3 - CD11c + MHCII + C CD11b + CD8 + C

13 3. nm UV 45 nm 488 nm 532 nm 561 nm 633/635 nm Alexa Fluor 35 442 Alexa Fluor 45 421 Pacific Blue 455 Pacific Green 5 Pacific Orange 551 Alexa Fluor 488 519 FITC 525 Qdot 65 65 Qdot 655 655 Qdot 75 75 Qdot 8 8 PerCP 678 PerCP-Cy5.5 695 R- R-PE, PE 575 PE-Texas Red 615 PE-Alexa Fluor 61 628 TRI-COLOR (TC, PE-Cy5) 67 PE-Cy 5.5 694 PE-Alexa Fluor 7 723 PE-Cy 7 767 APC 66 APC-Cy 5.5 694 APC-Cy 7 767 APC-Alexa Fluor 75 775 Alexa Fluor 647 668 Alexa Fluor 7 719 APC-Alexa Fluor 75 775 4. T 14 nm MHCII Pacific Blue dye 455 A1491 CD4 Pacific Green dye 5 C1127 B22 Pacific Orange dye 551 RM263 CD11b FITC 525 RM281 Foxp3 R-PE 575 N/A CD3 PerCP-Cy 5.5 695 A14784 CD11c PE-Cy 7 767 A15849 CD25 (IL-2R ) APC 66 N/A CD45.2 APC-Cy 7 767 A18642 CD8 Alexa Fluor 7 719 MCD829 * N/A

蛍光シグナル Qdot 45 nm 488 nm Qdot R-PE Qdot UV Qdot 45 nm Qdot 15 5 A B 吸光係数 (cm 1 M 1 ) 5,, 4,, 3,, 2,, 4 3 7 6 58 2 1,, 1 4 45 1. Qdot 525 2. Qdot 565 3. Qdot 585 4. Qdot 65 5. Qdot 625 6. Qdot 655 7. Qdot 75 8. Qdot 8 5 55 6 65 7 75 8 波長 ( nm) 1 2 3 4 5 6 7 8 45 5 55 6 65 7 75 8 85 9 波長 ( nm) Qdot www.thermofisher.com/ flow-qdot 15. Qdot A Qdot B Qdot A 5. Qdot 14 * Qdot 65 Qdot 655 Qdot 75 Qdot 8 CD2 S5.5 Q1172 Q2214 Q22141 Q22142 CD3e S4.1 (AKA 7D6) Q112 Q22149 Q1484 Q154 CD4 S3.5 Q18 Q17 _Q16 Q22153 S3.5 Q148 Q1482 Q1485 CD4 RM4-5 Q192 Q22163 Q22164 Q22165 CD8 3B5 Q19 Q155 Q159 Q22157 3B5 Q1481 Q1483 CD1 MEM-78 Q1481 " Q22136 CD14 HIT2 Q153 Q164 TüK4 Q113 Q156 Q22137 Q22139 CD19 SJ25-C1 Q136 Q1179 Q22138 Q22162 CD19 6D5 Q2216 Q1379 Q22161 CD27 CLB-27/1 Q165 Q166 Q22152 CD38 HIT2 Q2215 Q1156 CD45 H13 Q151 Q22154 Q162 Q22168 CD45R RA3-6B2 Q22166 Q1176 Q22167 Q22156 CD45RA MEM-56 Q147 Q169 Q22155 Q163 HLADR II Tü36 Q152 Q22158 Q22159 IgG2a Q114 Q115 Q176 * 1 25

15 mcherry mcherry 6 mcherry mcherry 16 mcherry IgG2a mcherry mcherry 6. mcherry 16D7 7 6 5 4 3 2 1 1 2 1 3 1 4 1 5 1 6 Pacific Blue fluorescence / nm Pacific Blue dye 45/455 M11238 Alexa Fluor 488 488/519 M11239 Alexa Fluor 594 59/617 M1124 Alexa Fluor 647 65/668 M11241 16. mcherry Pacific Blue mcherry M11238 U2-OS Attune Acoustic Focusing Cytometer 45 nm 522/31 nm Alexa Fluor Qdot R- R-PE 1 µg 1 mg IgG 2 2 www.thermofisher. com/flow-antibodylabeling SiteClick 1 µg IgG R-PE/Qdot 抗体 ポリクローナル抗体 モノクローナル抗体 組み換え抗体 N 3 N 3 N 3 N 3 アジド活性化抗体 β- ガラクトシダーゼ GalT(Y289L) UDP-GalNAz DIBO アルキン標識 Invitrogen TM SiteClick TM N SiteClick SiteClick 17 部位選択的に標識された抗体 17. SiteClick - N GlcNAc GlcNAc GalT Y289L GalNAz GlcNAc DIBO Alexa Fluor 488 DIBO alkyne 1 3 3.5

16 R-PE Qdot 7 SiteClick 18 SiteClick TM www.thermofisher.com/flow-siteclick Zenon Zenon IgG1 IgG2a IgG2b IgG Zenon 1 1% 1 1 Zenon Fab IgG Fc Zenon Fc Zenon Zenon 8 Zenon www.thermofisher.com/flow-antibodylabeling CD4 陽性細胞 (%) 6 4 2 初心者熟練研究者市販品 :R-PE SiteClick Qdot 65 SiteClick R-PE 18. SiteClick Qdot 65 Antibody Labeling Kit SiteClick R-PE Antibody Labeling Kit CD4 3 CD4 R-PE CD4 CD4 3 7. SiteClick nm SiteClick R-PE Antibody Labeling Kit 578 S1467 SiteClick Qdot 525 Antibody Labeling Kit UV 525 S1449 SiteClick Qdot 565 Antibody Labeling Kit UV 565 S145 SiteClick Qdot 585 Antibody Labeling Kit UV 585 S1451 SiteClick Qdot 65 Antibody Labeling Kit UV 65 S1469 SiteClick Qdot 625 Antibody Labeling Kit UV 625 S1452 SiteClick Qdot 655 Antibody Labeling Kit UV 655 S1453 SiteClick Qdot 75 Antibody Labeling Kit UV 75 S1454 SiteClick Qdot 8 Antibody Labeling Kit UV 8 S1455

17 8. Zenon Ex/Em* IgG1 IgG2a IgG2b IgG IgG IgG Alexa Fluor Alexa Fluor 35 346/442 Z25 Z251 Z253 Alexa Fluor 45 42/421 Z2513 Z25113 Z25213 Z25313 Alexa Fluor 488 495/519 Z252 Z2512 Z2522 Z2532 Z2562 Z2542 Alexa Fluor 546 556/573 Z254 Z2514 Z2534 Alexa Fluor 555 555/565 Z255 Z2515 Z2525 Z2535 Z2565 Alexa Fluor 568 578/63 Z256 Z2516 Z2536 Alexa Fluor 594 59/617 Z257 Z2517 Z2527 Z2537 Z2567 Z2547 Alexa Fluor 647 65/668 Z258 Z2518 Z2528 Z2538 Z2568 Z2548 Alexa Fluor 68 679/72 Z251 Z2511 Z2521 Alexa Fluor 7 696/719 Z2511 Alexa Fluor 75 749/775 Z25312 Pacific Blue 41/455 Z2541 Z25156 Pacific Green 411/5 Z1123 Pacific Orange 4/551 Z25256 494/518 Z2542 Z25342 XX NA Z2552 Z25152 Z25252 Z25352 Z25452 R-PE 496 /578 Z2555 Z25155 Z25255 Z25355 Z25455 R-PE-Alexa Fluor 61 496 /63 Z252 R-PE-Alexa Fluor 647 496 /668 Z2521 APC 65/66 Z2551 Z25151 Z25251 Z25351 Z25451 APC-zAlexa Fluor 75 65/775 Z2531 horseradish peroxidase; HRP NA Z2554 Z25154 Z25254 Z25354 Z25454 * nm Alexa Fluor Zenon 5 1 Zenon 1µg Zenon 25 1 546 nm 565 nm N/A www.thermofisher.com/flow-zenon

Alexa Fluor Alexa Fluor 19 Qdot 2 9 IgG www.thermofisher.com/flow-secondarydetection 9. Alexa Fluor 45 Pacific Blue Pacific Green Pacific Orange Alexa Fluor 488 PE Alexa Fluor 61 R-PE Alexa Fluor 647 R-PE APC Alexa Fluor 75 APC IgG A31553 P1993 P1124 A111 P852 A298 A299 A865 A216 IgG A31556 P1994 P31584 A118 P2771MP A2981 A2991 A1931 IgG A116 A1545 A154 S32351 S11222 S112 S32365 S11223 S866 S2982 S2992 S868 S32362* S218 * 18 A 1 B 4 1 C 4 1 4 橙色蛍光 (CD4) 1 3 1 2 1 1 赤色蛍光 (CD3) 1 3 1 2 1 1 赤色蛍光 (CD3) 1 3 1 2 1 1 1 1 1 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 緑色蛍光 (CD8) 橙色蛍光 (CD4) 緑色蛍光 (CD8) 1 4 19. Alexa Fluor 61 R-Phycoerythrin Tandem Streptavidin Conjugate Alexa Fluor488 R- 3 CD3 1% BSA PBSAlexa Fluor 61 R-Phycoerythrin Tandem Dye labeled Streptavidin S2982 Alexa Fluor 488 CD8 PE CD4 1% BSA/PBS Becton Dickinson FACScan 488 nm 525 + 1 nm CD8 575 + 1 nm CD4>65 nm CD3 A CD4/CD8 B CD3/CD4 C CD3/CD8

19 RUO jpcustom@lifetech.com 製品紹介 結果まで約 1 秒 細胞カウントが楽しくなる ess Ⅱ FL ess Ⅱ 自動セルカウンター Invitrogen TM ess TM Ⅱ FL ess TM Ⅱ 自動セルカウンターは 細胞カウントを迅速かつ正確に行うことが可能です タッチスクリーンでの直観的な操作 約 1 秒の正確な細胞数のカウント オートフォーカス機能による実験誤差の最小化で手間を大幅に削減します 取得したイメージ画像データは画面上で見ることができ 計測が正確に行われているか確認して次の実験に進むことができ 安心です

www.thermofisher.com/flow-cellhealth A 125 1 未固定 生細胞 死細胞 75 5 2 1 www. thermofisher.com/flow-cellviability B 25 1 2 1 3 1 4 1 5 LIVE/DEAD Fixable Aqua fluorescence 15 固定処理後死細胞 LIVE/DEAD Fixable Dead Cell Stain Kits UV/45/488/633 7 1 5 生細胞 LIVE/DEAD Fixable Dead Cell Stain Kit 3 5 2 LIVE/DEAD Fixable Dead Cell Stain LIVE/DEAD Fixable Dead Cell Stain ArC Amine Reactive Compensation Bead Kit A1346 8 1 2 1 3 1 4 1 5 LIVE/DEAD Fixable Aqua fluorescence 2. LIVE/DEAD Fixable Dead Cell Stains Jurkat 2 LIVE/ DEAD Fixable Aqua Dead Cell Stain Kit L34957 A B 3.7% 45 nm 525 nm 2 Perfetto 26 21 LIVE/DEAD Fixable Dead Cell Stain 1. * LIVE/DEAD Fixable Dead Cell Stains SYTOX Dead Cell stains Propidium lodide Ready Probes Reagent *

21 SYTOX Dead Cell Stains Invitrogen TM SYTOX TM Dead Cell Stains 3 DNA SYTOX 22 SYTOX Dead Cell Stains DNA リンパ球のゲーティング 生存率のゲーティング A B A C CD4 Cy 5.5-PE fluorescence FSC-A SSC CD8 Q75 fluorescence 21. A B LIVE/DEAD Fixable Violet Dead Cell Stain Kit L34955 C D 2 Perfetto SP, Chattopadhyay PK, Lamoreaux L, et al. (26) J Immunol Methods 313:199 28 Elsevier D CD4 Cy 5.5-PE fluorescence FSC-A 生細胞 死細胞 LIVE/DEAD Fixable Violet fluorescence CD8 Q75 fluorescence B CD3 Alexa Fluor 488 fluorescence 2 15 1 5 1 5 1 4 1 3 生細胞 死細胞 1 2 1 3 1 4 1 5 SYTOX Red fluorescence 1 3 1 4 1 5 CD8 R-PE fluorescence 22. SYTOX Dead Cell Stains Alexa Fluor 488 CD3 R-PE CD8 5 nm SYTOX Red Dead Cell Stain S34859 488 nm/635 nm A 2 SYTOX B CD8 CD3 2

Invitrogen TM CellTrace TM Cell Proliferation Kit DNA Invitrogen TM Click-iT TM Plus EdU 11 www.thermofisher.com/flow-cellproliferation Click-iT Plus EdU Flow Cytometry Assay Kits BrdU CV 5 GFP mcherr APC PerCP PE DNA EdU 5- -2 -DNA Click-iT Plus EdU Flow Cytometry Assay Kit EdU DNA DNA BrdU DNA BrdU BrdU DNase DNA Click-iT Plus EdU Flow Cytometry Assay DNA 23 Click-iT Plus EdU Click-iT Plus Kit Click-iT Click-iT Plus R-PE R-PE GFP Click-iT Plus EdU Click-iT EdU 3 22 A 2 B 9 C 8 15 7 6 5 1 4 3 5 2 1 1 1 1 2 1 3 1 4 1 5 1 6..5 1. 1.5 2. Click-iT EdU plus Alexa Fluor 488 FxCycle Violet fluorescence (x1 6 ) fluorescence Click-iT EdU plus Alexa Fluor 488 fluorescence 1 6 1 5 1 4 1 3 1 2..5 1. 1.5 2. FxCycle Violet fluorescence (x1 6 ) 23. Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit FxCycle Violet Stain Jurkat 1 µm EdU 1 Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit C1632 Alexa Fluor 488 FxCycle Violet Stain F1347 488 nm Click-iT EdU Alexa Fluor 488 45 nm FxCycle Violet Stain A S EdU DNA G2/M G/G1 BFxCycle Violet Stain DNA G/G1 G2/M S C Click-iT Plus EdU FxCycle 2 S

23 11. Click-iT Plus EdU Flow Cytometry Assay Kits DNA CellTrace Cell Proliferation Kits CellTrace Cell Proliferation Kits GFP CellTrace CellTrace CFSE Dye C34554 CellTrace Violet Dye C34557 CellTrace Far Red Dye C34564 3 CellTrace CellTrace CFSE CellTrace Violet 8 1 24 CellTrace CellTrace Violet CellTrace Far Red GFP A B C 4 1 1 3 2 8 6 4 8 6 4 1 2 2 1 2 1 3 1 4 1 5 1 6 1 3 1 4 1 5 1 6 1 3 1 4 1 5 1 6 CellTrace Violet fluorescence CellTrace CFSE fluorescence CellTrace Far Red fluorescence 24. CellTrace A CellTrace Violet 8 CellTrace Violet CD3 MHCD3 2 PHC27 7 CD4 B CellTrace CFSE C CellTrace Far Red 7 T CD3 MHCD3 5 SYTOX Green S3486CellTrace CFSE CellTrace Far RedAttune Acoustic Focusing Cytometer CellTrace Violet 45 nm 45/4 nm CellTrace CFSE 488 nm 53/3 nm CellTrace Far Red 638 nm 66/2 nm

SYTOX Blue fluorescence DNA G/G1 S G2/M 12 www. thermofisher.com/flow-cellcycle Vybrant DyeCycle Stains side population A B 1 5 1 4 死細胞 1 3 1 2 生細胞 5 1 15 2 25 Vybrant DyeCycle Green fluorescence 2, 1,5 1, 5 24 Invitrogen TM Vybrant TM DyeCycle TM Stains DNA 45 488 532 633 nm 25 Vybrant DyeCycle Stains 3 FxCycle Stains 488 nm Invitrogen TM FxCycle TM Violet Stain F1347 FxCycle PI/RNase Staining Solution F1797 FxCycle Far Red Stain F1348 22 23 FxCycle Stains DNA 12. 5 1 15 2 25 Vybrant DyeCycle Green fluorescence 25. Vybrant DyeCycle Stains Jurkat Vybrant DyeCycle Green Stain V354 SYTOX Blue Dead Cell Stain S34857 488 nm 45 nm A B DNA G/G1 G2/M S Vybrant DyeCycle Stains DNA FxCycle Stains DNA

25 13 www.thermofisher.com/flow-apoptosis CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit 3/7 Invitrogen TM CellEvent TM Caspase-3/7 Green Flow Cytometry Assay Kit C1427 3 7 DNA 26 A SYTOX ADDvanced fluorescence B SYTOX ADDvanced fluorescence 1 6 1 5 1 4 1 2 1 3 コントロール V 1 4 1 3 1 2 1 3 1 5 1 5 CellEvent Caspase-3/7 Green fluorescence 1 6 1 5 1 4 1 2 1 3 誘導 V 1 4 1 3 1 2 1 3 1 5 1 5 CellEvent Caspase-3/7 Green fluorescence 26. CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit Jurkat Jurkat T A DMSO B 1 µm 3 CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit C1427 488 nm Attune Acoustic Focusing Cytometer CellEvent 53/3 nm SYTOX AADvanced 69/5 nm B A A N V N N A A 13. CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit 3/7 1 V 1 MitoProbe JC-1 Assay Kit 2 Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit 3

JC-1 red fluorescence - BL-3 JC-1 red fluorescence - BL-3 26 V Invitrogen TM PS PS V PS Alexa Fluor 27 1 V 4 D 1 3 3 V 31 1 2 www.thermofisher.com/flow-annexin V MitoProbe JC-1 Assay Kit 488 nm 633/635 nm 1 1 1 1 1 2 1 3 1 4 Invitrogen TM MitoProbe TM JC-1 Assay Kit M34152 JC-1 3- CCCPDMSO PBSJC-1 J ~529 nm ~59 nm 28 A B ヨウ化プロピジウム蛍光 1 1 A A B ヨウ化プロピジウム蛍光 ヨウ化プロピジウム蛍光 1 4 1 3 1 2 1 1 V D 1 1 1 1 1 2 1 3 1 4 Alexa Fluor 488 fluorescence 1 4 1 3 1 2 1 1 V D 1 1 1 1 1 2 1 3 1 4 Alexa Fluor 488 fluorescence 27. Alexa Fluor 488 V ジ ヨウ化プロピジウム Jurkat ロ Jurkat T A 1 µm 4 B Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide V13245 A V D A A JC-1 蛍光 1 5 1 5 1 4 1 4 1 5 1 5 JC-1 蛍光 1 4 1 4 1 3 1 3 1 3 1 3 1 3 1 3 1 4 1 4 1 5 1 5 1 3 1 3 1 4 1 4 1 5 1 5 JC-1 蛍光 JC-1 蛍光 28. 2 µm JC-1 Jurkat MitoProbe JC-1 Assay Kit M3415237 C 5% CO2 2 PBS 488 nm Attune Acoustic Focusing Cytometer 53/3 nm >64 nm A B 1 µm 37 C 5

SYTOX AADvanced fluorescence SYTOX AADvanced fluorescence 27 5 Invitrogen TM Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit A35137 29 Violet Ratiometric Membrane Asymmetry Probe ESIPT 53 nm 585 nm 2 2 2 F2N12S SYTOX AADvanced Dead Cell Stain A 262,144 B 262,144 F2N12S 緑色蛍光 196,68 131,72 65,536 A+D F2N12S 緑色蛍光 196,68 131,72 65,536 A+D 65,536 131,72 196,68 262,144 F2N12S 橙色蛍光 L 65,536 131,72 196,68 262,144 F2N12S 橙色蛍光 L C 1 5 D D 1 5 D 1 4 1 4 1 3 A L 1 3 L A 1 2 1 2 1 1 1,25 2,5 3,75 5, F2N12S ratio 585 nm/53 nm fluorescence x 1, 1 1 1,25 2,5 3,75 5, F2N12S ratio 585 nm/53 nm fluorescence x 1, 29. Violet Ratiometric Membrane Asymmetry Probe Jurkat T 1 µm 4 B D A CF2N12S 45 nm 585 nm 53 nm SYTOX AADvanced Dead Cell Stain 488 nm 695 nm F2N12S 2 A B C D F2N12S 2 585/53 nm SYTOX AADvanced Dead Cell Stain A = L = D =

SYTOX Blue fluorescence SYTOX Blue fluorescence CellROX Flow Cytometry Kits ROS ROS ROS Invitrogen TM CellROX TM 3 CellROX Green Flow Cytometry Assay Kit C1492 CellROX Orange Flow Cytometry Assay Kit C1493 CellROX Deep Red Flow Cytometry Assay Kit C1491 3 www.thermofisher.com/flow-cellrox A B C 1 5 死細胞 3.1% ++.21% 1 5 死細胞 4.74% ++ 1.25% 1 4 1 4 1 3 1 3 12 1 3 1 4 1 5 CellROX Deep Red fluorescence 1 2 生細胞 96.1% ROS+.852% 1 2 1 3 1 4 1 5 CellROX Deep Red fluorescence 1 2 生細胞.91% ROS+ 93.1% 1 2 1 3 1 4 1 5 CellROX Deep Red fluorescence 28 3. ROS A CellROX Deep Red Flow Cytometry Assay Kit C1491 CellROX Deep Red Reagent Jurkat ROS N- NAC ROS tert- TBHP NAC TBHP B C CellROX Deep Red Reagent SYTOX Blue Dead Cell Stain Jurkat B PBS C 2 µm TBHP 3 CellROX Deep Red Flow Cytometry Assay Kit TBHP C ROS B

29 phrodo E. coli BioParticles Phagocytosis Kits phrodo phrodo ph phrodo ph ph Invitrogen TM phrodo TM Green E. coli BioParticles TM Phagocytosis Kit P35381 phrodo Red E. coli BioParticles Phagocytosis Kit A125 31 phrodo Phagocytosis Particle Labeling Kit A126 phrodo Red www.thermofisher.com/flow-phrodo 12 8 ネガティブコントロール 4 ファゴサイトーシスされた粒子 1 1 1 1 2 1 3 1 4 phrodo dye fluorescence (585 nm) 31. phrodo Red BioParticles 1 µl 2 phrodo Red E. coli BioParticles Phagocytosis Kit A125 phrodo Red E. coli BioParticles 2 37 C 15 2 5 x g 5 488 nm BD FACSCalibur Cytometer 564 66 nm 37 C phrodo Red BioParticles

3 Ex/Em* LIVE/DEAD Fixable Blue Dead Cell Stain Kit UV 35/45 2 L2315 LIVE/DEAD Fixable Violet Dead Cell Stain Kit 416/451 2 L34955 LIVE/DEAD Fixable Aqua Dead Cell Stain Kit 367/526 2 L34957 LIVE/DEAD Fixable Yellow Dead Cell Stain Kit 4/575 2 L34959 LIVE/DEAD Fixable Green Dead Cell Stain Kit 495/52 2 L2311 LIVE/DEAD Fixable Red Dead Cell Stain Kit 595/615 2 L2312 LIVE/DEAD Fixable Far Red Dead Cell Stain Kit LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit LIVE/DEAD Fixable Dead Cell Stain Sampler Kit SYTOX Blue Dead Cell Stain 65/665 2 L112 75/775 2 L1119 32 L3496 UV/ 444/48 1 ml S34857 SYTOX Green Dead Cell Stain 54/523 1 ml S3486 SYTOX Orange Dead Cell Stain 547/57 1 ml S34861 SYTOX Red Dead Cell Stain 64/658 1 ml S34859 SYTOX Dead Cell Stain Sampler Kit 1 S34862 SYTOX AADvanced Dead Cell Stain 546/647.2 ml S1349 5 x.5 ml S1274 Click-iT Plus EdU Pacific Blue Flow Cytometry Assay Kit 44/45 5 C1636 Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit 495/519 Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit 65/67 5 C1632 1 C1633 5 C1634 1 C1635 CellTrace Violet Cell Proliferation Kit 45/45 18 C34557 CellTrace CFSE Cell Proliferation Kit 492/517 1 C34554 CellTrace Far Red Cell Proliferation Kit 63/661 1 C34564 Vybrant DyeCycle Violet Stain 369/437 2 µl V353 Vybrant DyeCycle Green Stain 56/534 4 µl V354 Vybrant DyeCycle Orange Stain 519/563 4 µl V355 Vybrant DyeCycle Ruby Stain 638/686 4 V1273 1 V139 FxCycle Violet Stain 358/461 5 F1347 FxCycle PI/RNase Staining Solution 535/617 1 ml F1797 FxCycle Far Red Stain 64 658 5 F1348 * Ex Em nm DNA / nm J

31 CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit / Ex/Em* 511/533 (CellEvent Green) 546/647 1 C1427 (SYTOX AADvanced Stain) Annexin V, Pacific Blue conjugate 41/455 5 µl A35122 Annexin V, FITC conjugate 494/518 5 µl A13199 Annexin V, Alexa Fluor 488 conjugate 495/519 5 µl A1321 Annexin V, PE conjugate 496, 546, 565/578 25 µl A35111 Annexin V, APC conjugate 65/66 25 µl A3511 Annexin V, Alexa Fluor 647 conjugate 65/665 5 µl A2324 Pacific Blue Annexin V/SYTOX ADvanced Apoptosis Kit Dead Cell Apoptosis Kit with Alexa Fluor 488 Annexin V & Propidium Iodide Dead Cell Apoptosis Kit with FITC annexin V & Propidium Iodide Violet Ratiometric Asymmetry Probe/Dead Cell Apoptosis Kit / / 41/455 Pacific Blue dye 546/647 (SYTOX AADvanced dye) 495/519 (Alexa Fluor 488 dye) 5 A35136 5 V13241 535/617 (PI) 25 V13245 494/519 (FITC) 535/617 (PI) 45 53, 585 (F2N12S) 546/647 (SYTOX AADvanced dye) 5 V13242 1 A35137 MitoProbe JC-1 Assay Kit 514/529, 59 1 M34152 CellROX Green Flow Cytometry Assay Kit CellROX Orange Flow Cytometry Assay Kit CellROX Deep Red Flow Cytometry Assay Kit phrodo Green E. coli BioParticles Phagocytosis Kit phrodo Green S. aureus BioParticles Phagocytosis Kit phrodo Red E. coli BioParticles Phagocytosis Kit phrodo Phagocytosis Particle Labeling Kit 532 nm 58/525 (CellROX Green) 64/658 1 C1492 (SYTOX Red) 545/565 (CellROX Orange) 64/658 1 C1493 (SYTOX Red) 64/665 (CellROX Deep Red) 444/48 1 C1491 (SYTOX Blue) 59/533 1 P35381 59/533 1 P35382 56/585 1 A125 56/585 1 A126 * Ex Em nm DNA / nm J

4 14 Attune NxT Acoustic Focusing Cytometer Invitrogen TM Attune TM NxT Acoustic Focusing Cytometer 1 1 4 14 1 5 cells /ml 1, events.5 5 µm = OFF = ON Attune NxT cytometer 14-color laser configurations 44/5 Pacific Blue ECFP Violet 45 nm 512/25 Pacific Green 63/48 Pacific Orange 71/5 Qdot 75 NA FSC NA SSC*** 53/3 FITC EGFP, Emerald GFP Blue 488 nm 574/26* PE EYFP 59/4** PE, PE Texas Red 695/4 PerCP-Cy5.5 78/6* PE-Cy7 585/16 PE RFP Yellow 561 nm 62/15 PE-Texas Red mcherry, tdtomato, DsRed, mstrawberry 695/4 PE-Cy 5.5 78/6 PE-Cy 7 67/14 APC Red 638 nm 72/3 Alexa Fluor 7 78/6 APC-Alexa Fluor 75 * Yellow ** Yellow *** / 32

4 cm 58 cm 43 cm 29 kg 15 3 1 9% 1 24 VAC 5/6 Hz <15 W 1. m 65 dba 1 4 :Blue laser: 488 nm, 5 mw Violet laser: 45 nm, 5 mw Red laser: 637 nm, 1 mw Yellow laser: 561 nm, 5 mw : : 1.2NA :, 488/1 488/1 PMT : 12.5 1, µl/min : : 2 µl 4 ml : : 1.8 L, 1.8 L, 175 ml, 175 ml 1 L : 1 1.8 L : 17 1 mm 8.5 45 mm Levey-jennings 15 2 SmartGate Quad Stats math calculator Stain Index S / N FCS3.1 35, /.5 5 µm 8 MESF FITC 3 MESF PE 7 MESF APC Attune NxT Autosampler 96/384 <1% 33

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