Cell Counting Kit p. 3 Cell Counting Kit -8 MTT p. 12 Cell Counting Kit -F p. 19 p. 26 -Cellstain- Calcein-AM -Cellstain- CFSE -Cellstain- Calcein-AM solution -Cellstain- CytoRed solution -Cellstain- FDA BCECF-AM special packaging -Cellstain- DAPI -Cellstain- EB -Cellstain- PI -Cellstain- DAPI solution -Cellstain- EB solution -Cellstain- PI solution p. 28 -Cellstain- AO -Cellstain- AO solution -Cellstain- Hoechst 33258 solution -Cellstain- Hoechst 33342 solution -Cellstain- MitoRed -Cellstain- Rh123 -Cellstain- Double Staining Kit p. 37
3 H MTT WST 3 H RI DNA RI MTT WST 19
Cell Counting Kit-8 Cell Counting Kit 8 Cell Counting Kit Cell Counting Kit-8 WST-1 (Cell Counting Kit) WST-8 (Cell Counting Kit-8) MTT 3 High-Throughput Screening WST Cell Counting Kit CellCounting Kit-8 96 Cell Counting Kit Cell Counting Kit-8 Cell Counting Kit : 400 450 nm Cell Counting Kit-8 : 450 490 nm 96 8 12 10 100 Cell Counting Kit CK01 11632074 1025 Cell Counting Kit -8CK04 Cell Counting Kit A B!! 4 12 4 3 20 1-20 Cell Counting Kit-8!! 4 12-20
Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit-8 cells /ml 96 100 24 48 96 Cell Counting Kit Cell Counting Kit-8 10 1 4 96 1/10 Cell Counting Kit : 400 450 nm Cell Counting Kit-8 : 450 490 nm HeLa HL60
Cell Counting Kit-8 Cell Counting Kit HeLa HL60 96 2.5 10 4, 1.25 10 4, 6.2 10 3,0 cells well Cell Counting Kit-8 8 96 100 5 10 5 cells/ml 100 100 HeLa HL60 2.5 HeLa 0.5 HL60 2.0 0.4 Absorbance 1.5 1.0 Absorbance 0.3 0.2 0.5 0.1 0.0 0 5000 10000 15000 20000 25000 30000 0.0 0 5000 10000 15000 20000 25000 30000 Number of cells (/well) Number of cells (/well) : DMEM (10% FBS) : RPMI1640 (10% FBS) 37, 3 hr, 37, 3.5 hr, 5% CO 2 5% CO 2 : 450 nm : 450 nm Cell Counting Kit-8 [ 3 H]- HeLa Cell Counting Kit-8 [ 3 H]- Absorbance 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.5 1.0 1.5 2.0 [ 3 H]-Thymidine x 10-5 dpm
Cell Counting Kit-8 Cell Counting Kit cells /ml 96 100 24 48 48 100 10 10 6 12 24 48
Cell Counting Kit-8 Cell Counting Kit 96 Cell Counting Kit 96 Cell Counting Kit-8 10 1/10 1 4 450 nm n =5 A sample A sample - A blank 100 A control - A blank A sample 1 A sample 2 Cell Counting Kit Cell Counting Kit-8 A sample 3 A sample 4 A sample 5 A sample n Cell Counting Kit Cell Counting Kit-8 A control Cell Counting Kit Cell Counting Kit-8 A blank Cell Counting Kit, Cell Counting Kit-8 WST-1 WST-8 Cell Counting Kit Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit-8 PBS(-)
Cell Counting Kit-8 Cell Counting Kit 8 8 17 8 1 w /v 10
Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit (Cell Counting Kit-8) WST-1, WST-8 O2N O2N O2N H N N N N WST-8 formazan N N N N OCH3 SO3- SO3- SO3- SO3-1-Methoxy PMS 1-Methoxy PMS ( 還 元 体 ) 脱 水 素 酵 素 NADH, NADPH NAD +, NADP + 酸 化 体 基 質 OCH3 Na+ O2N WST-8 WST-1(WST-8) 1-Methoxy PMS WST-1(WST-8) 1-Methoxy PMS Cell Counting Kit (Cell Counting Kit-8) MTT Cell Counting Kit (Cell Counting Kit-8) 24 90% MTT 0% Cell Counting Kit (Cell Counting Kit-8) Cell Counting Kit (Cell Counting Kit-8) 12 48 Cell Counting Kit (Cell Counting Kit-8) Cell Counting Kit (Cell Counting Kit-8) 96 1000 10000
Cell Counting Kit-8 Cell Counting Kit 24 well 12 well 96 well 10 1 1 ml 100 96 well 1 w/v% SDS 10 0.1 mol/ l (10 ) 24 1 4 Cell Counting Kit (Cell Counting Kit-8) 1 8 5 600 nm 1 8 600 nm 1 8 450 nm 8 8450 nm 1 8 8450 nm 1 8
Cell Line Description Reference 293T human kidney carcinoma H. Fuda, et al., J. Lipid Res., 2007, 48, 1343. 3T3-L1 mouse embryonic fibroblast D. Huang, et al., FASEB J., 2005, 19, 2014. A431 human epithelial carcinoma cell H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. A549 human lung cancer cell C. A. Reilly, et al., Toxicol. Sci., 2003, 73, 170. ; T. Kitamuro, et al., J. Biol. Chem., 2003, 278, 9125. J. Stankova, et al., Clin. Cancer Res., 2005, 11, 2047. ;I.Imoto, et al., Cancer Res., 2006, 66, 4617. ; S. Semba, et al., J. Biol. Chem., 2006, 281, 28244. Alexander cell pancreatic cancer cell S. Awale, et al., Cancer Res., 2006, 66, 1751. AMO1 multiple myeloma J. Inoue, et al., Am. J. Pathol., 2004, 165, 71. AR42J pancreatic tumor cell C. Bose, et al., Am. J. Physiol. Gastrointest. Liver Physiol., 2005, 289, G926. ARO human anaplastic thyroid carcinoma F. Furuya, et al., Endocrinology, 2004, 145, 2865. AsPC-1 pancreatic cancer cell T. Mori, et al., Mol. Cancer Ther., 2004, 3, 29. ; S. Awale, et al., Cancer Res., 2006, 66, 1751. B16F1 murine malignant melanoma S. Shibata, et al., J. Immunol., 2006, 177, 3564. BBMVEC bovine brain microvascular endothelial T. Kitamuro, et al., J. Biol. Chem., 2003, 278, 9125. BEAS-2B human bronchial epithelial cell C. A. Reilly, et al., Toxicol. Sci., 2003, 73, 170. ; M. E. Johansen, et al., Toxicol. Sci., 2006, 89, 278. BMMSC bone marrow mesenchymal stem cell M. Miura, et al., Stem Cells, 2006, 24, 1095. C33A human cervical carcinoma W. Yang, et al., Mol. Cancer Ther., 2006, 5, 1610. CHO cortical neurons, primary COS7 chinese hamster ovary cell A.Kunita, et al., Am. J. Pathol., 2007, 170, 1337.; S. Yokoe, et al., Cancer Res., 2007, 67, 1935. mouse M. Ikonen, et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 13042. T. Kitamuro, et al., J. Biol. Chem., 2003, 278, 9125. ; H. Bando, et al., Clin. Cancer Res., 2005, 11, 5784. African green monkey kidney cell CV-1 African green monkey kidney cell J. Peloponese, et al., J. Biol. Chem., 2006, 281, 8927. Daoy human medulloblastoma X. Li, et al., Mol. Cancer Ther., 2005, 4, 1912.; S. Kim, et al., Clin. Cancer Res., 2006, 12, 5550. Daudi human burkitt lymphoma M. Ho, et al., J. Biol. Chem., 2005, 280, 607. DLD-1 human colorectal adenocarcinoma H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. DT40 chicken B-lymphocyte cell F. Shinozaki, et al., J. Biol. Chem., 2006, 281, 16361. DU145 human prostate carcinoma P. Davis-Searles, et al., Cancer Res., 2005, 65, 4448. ; D. J. Son, et al., Mol. Cancer Ther., 2007, 6, 675. G361 human melanoma cell H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. GCIY human gastric carcinoma H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. H441 human pulmonary adenocarcinoma H. Shimura, et al., Cancer res., 2001, 61, 3640. HCC1937 human breast cancer cell D. Iliopoulos, et al., Clin. Cancer Res., 2007, 13, 268. HEK293 human embryonic kidney cell S. Semba, et al., J. Biol. Chem., 2006, 281, 28244. H. Shimura, et al., Cancer res., 2001, 61, 3640.; C. Shi, et al., J. Biol. Chem., 2004, 279, 17224. HeLa human cervical carcinoma Hep3B human hepatocellular carcinoma J. K. Sicklick, et al., Carcinogenesis, 2006, 27, 748. HepG2 Hippocampal neuron, primary hmsc Human hepatocellular liver carcinoma C. A. Reilly, et al., Toxicol. Sci., 2003, 73, 170.; T. Ohuchida, et al., Cancer, 2004, 100, 2430.; T. Shiokawa, et al., Clin. Cancer Res., 2005, 11, 2018. ; H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. from Wistar rat embryos K. Kurata, et al., J. Pharmacol. Exp. Ther., 2004, 311, 237. D. Huang, et al., FASEB J., 2005, 19, 2014. ;L. Song, et al., Stem Cells, 2006, 24, 1707. human mesenchymal stem cell ILT-Hod T-cell T. Kasai, et al., J. Biol. Chem., 2002, 277, 5187. Jurkat human T cell Kasumi-1 acute myeloid leukemia cell G. Zhou, et al., Blood, 2007, 109, 3441. KMS-11 multiple myeloma J. Inoue, et al., Am. J. Pathol., 2004, 165, 71. KYSE esophageal squamous cell carcinoma T. Kasai, et al., J. Biol. Chem., 2002, 277, 5187. ; L. Lu, et al., J. Biochem., 2007, 141, 157. I. Imoto, et al., Cancer Res., 2001, 61, 6629. ;K. Nakakuki, et al., Carcinogenesis, 2002, 23, 19. L929 mouse fibroblast Y. Morita, et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5405. LK87 human lung adenocarcinoma H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. LNCaP human prostate carcinoma D. J. Son, et al., Mol. Cancer Ther., 2007, 6, 675. Macrophage mouse Y. Miyake, et al., J. Immunol., 2007, 178, 5001. MDCK canine kidney epithelial cell H. Shimura, et al., Cancer res., 2001, 61, 3640. MiaPaCa-2 pancreatic cancer cell A. Aghdassi, et al., Cancer Res., 2007, 67, 616. T. Kasai, et al., J. Biol. Chem., 2002, 277, 5187; K. V. Kibler, et al., J. Biol. Chem., 2004, 279, 49055. MT4 T-cell NIH3T3 mouse fibroblast R. Yu, et al., Toxicol. Sci., 2006, 93, 82. NT2N human embryonal carcinoma J. Tessier, et al., Infect. Immun., 2007, 75, 1895. PANC-1 pancreatic cancer cell T. Mori, et al., Mol. Cancer Ther., 2004, 3, 29.; S. Awale, et al., Cancer Res., 2006, 66, 1751; A. Aghdassi, et al., Cancer Res., 2007, 67, 616. PC6 human lung small-cell carcinoma M. Spencer, et al., J. Biol. Chem., 2002, 277, 20160. PCN primary cortical neuron T. Chiba, et al., J. Neurosci., 2005, 25, 10252. PSN-1 pancreatic cancer cell S. Awale, et al., Cancer Res., 2006, 66, 1751. RAW 264 mouse macrophage M. Shiga, et al., Anesth. Analg., 2001, 92, 128. RP9 B lymphoblastoid cell R. A. Dalloul, et al., Poult. Sci., 2006, 85, 446. SH10TC human gastric cancer cell H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 2563. SH-SY5Y human neuroblastoma T. Nakagawa, et al., Mol. Cell. Biol., 2002, 22, 2575. SK-N-SH human neuroblastoma Y. Wang, et al., J. Virol., 2004, 78, 7916.
MTT MTT Cell Counting Kit [ 3 H] MTT 570 535 DMSO 96 8 12 10 100 MTTM009 PBS(-) DMSO PBS(-)Ca 2+, Mg 2+ MTT MTT 25 mg PBS(-) 5 ml! 0.22! -20! 4 mol/l 0.1 ml 10 ml 4! mol/l
MTT MTT MTT cells /ml 96 100 24 48 96 MTT 10 2 4 PBS(-) 200 1 96 1/10 DMSO 200 570 DMSO535
MTT HeLa 96 2.5 10 4, 1.25 10 4, 6.2 10 3,0 cellswell MTT 8 96 100 5 10 5 cells/ml 100 100 24 MTT DMSO DMSO MTT 570DMSO 535 nm MTT MTT MTT Cell Counting Kit, Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit-8 WST-1WST-8 Cell Counting Kit Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit-8
MTT cells /ml 96 100 24 48 48 100 10 10 l 6 12 24 48 96 96 MTT 10 1/10
MTT 2 4 PBS() 200 1 MTT 200 570 535 n =5 A sample A sample - A blank A control - A blank 100 A sample 1 A sample 2 MTT A sample 3 A sample 4 A sample 5 A sample n MTT A control MTT A blank
MTT 650 nm MTT MTT MTT 2 4 MTT DMSO MTT MTT
MTT MTT AMTT MTT ミトコンドリア 脱 水 素 酵 素 H H3C N N N N N S H3C N N N N N S CH3 CH3 黄 色 溶 液 MTT 難 溶 性 沈 殿 MTT formazan / DMSO MTT / DMSO MTT
Cell Counting Kit-F Cell Counting Kit-F Cell Counting Kit MTT Calcein-AM Calcein Cell Counting Kit Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit-8 480 500 nm 500 535 nm 96 8 12 10 100 Cell Counting Kit -F06 PBS(-)Ca 2+, Mg 2+! 12120 Cell Counting Kit-F Cell Counting Kit-F PBS(-) 50!
Cell Counting Kit-F Cell Counting Kit-F Cell Counting Kit-F cells/ml 96 100 24 48 PBS(-) 100 V 96 Cell Counting Kit-F 96 1/10 10 15 30 480 500 nm 500 535 nm
Cell Counting Kit-F HeLa HL60 96 2.5 10 4, 1.25 10 4, 6.2 10 3,0 cellswell Cell Counting Kit-F 8 96 100 5 10 5 cells/ml 100 100 HeLa HL60 Cell Counting Kit-F 40000 HeLa 40000 HL60 Relative Fluorescence Intensity 30000 20000 10000 Intensity Relative Fluorescence Intensity 30000 20000 10000 Intensity 0 0 5 10 15 20 25 Number of Cells (x10 3 cells/well) 0 250 500 750 1000 Number of Cells 0 0 5 10 15 20 25 Number of Cells (x10 3 cells/well) 0 250 500 750 1000 Number of Cells
Cell Counting Kit-F cells /ml 96 100 24 48 48 100 10 10 6 12 24 48
Cell Counting Kit-F PBS(-) 100 96 Cell Counting Kit-F 10 15 30 96 1/10 480 500 nm 500 535 nm n =5 A sample A sample - A blank 100 A control - A blank A sample 1 A sample 2 Cell Counting Kit-F A sample 3 A sample 4 A sample 5 A sample n Cell Counting Kit-F A control Cell Counting Kit-F A blank
Cell Counting Kit-F Calcein-AM Cell Counting Kit-F Calcein-AM) PBS(-) Cell Counting Kit-F Cell Counting Kit-F Cell Counting Kit-F
Cell Counting Kit-F Cell Counting Kit-F Calcein-AM (Calcein) Calcein Calcein-AM Calcein-AM ph ph Calcein-AM Calcein Calcein Cell Counting Kit-F Calcein-AM Cell Counting Kit-F Cell Counting Kit-8 Cell Counting Kit-F Calcein-AM Calcein Calcein ROOC ROOC N N COOR COOR esterase HOOC HOOC N N COOH COOH H3COCO O OCOCH3 HO O O O O 無 蛍 光 R=CH2OCOCH3 O HO 蛍 光
MTT WST-1, WST-8, XTT, MTS NADH NADH ATP ATP ATP - - - - 10 MTT, WSTCalcein-AM ATP ATP ATP ATP ATP Cell Lysis ATP ATP ATP ATP ATP ATP ATP + D-luciferin + O 2 ATP oxyluciferin + PPi + AMP + CO 2 + light
DNA [ 3 H]- [ 3 H]-DNA BrdU BrdUDNABrdUBrdU C-G A-T A-T T-A C-G C- -G A- -T G- -C A- A- -T C-G A-T A-T T-A C-G C-G A-T G-C A-T A-T -T or -U [ 3 H]- BrdU [ 3 H]-BrdU DNA [ 3 H]-BrdU (LDH) - - - - WSTNK LDH LDH LDH Lactate Pyruvate LDH LDH INT formazan or NAD+ Resorufin Diaphorase NADH INT or Rezazurin LDH
Calcein-AMFluorescein diacetate (FDA)CFSE fluorescein Propidium iodide (PI)Ethidium bromide (EB) DAPI DNA
-Cellstain- Calcein-AM -Cellstain- CFSE -Cellstain- FDA -Cellstain- DAPI -Cellstain- EB -Cellstain- PI -Cellstain- Calcein-AM solution -Cellstain- CytoRed solution BCECF-AM -Cellstain- DAPI solution -Cellstain- EB solution -Cellstain- PI solution DMSO PBS(-) -Cellstain- AO -Cellstain-AO solution -Cellstain- Hoechst 33258 -Cellstain- Hoechst 33342 -Cellstain- MitoRed -Cellstain- Rh123 ( DMSO (DMSO ) -Cellstain- Calcein-AM 994.86 1 mg 0.5-1mmol/l -Cellstain- Calcein-AM solution 994.86 1 ml 1mmol/l 1-20 mol/l -Cellstain- CFSE 557.64 1 mg 0.5-1mmol/l ( PBS(-) -Cellstain- CytoRed solution 313.31 1 ml 1mmol/l ) -Cellstain- FDA 416.38 1 mg 0.5-1mmol/l BCECF-AM Special packging 688.59 50 µg x 8 0.5-1mmol/l! PBS(-)
-Cellstain- DAPI 350.25 1 mg 1 mg/ml -Cellstain- DAPI solution 350.25 1 ml 1 mg/ml of Buffer μ -Cellstain- EB 394.31 1 mg 1 mg/ml -Cellstain- EB solution 394.31 1 ml 1 mg/ml -Cellstain- PI 668.39 1 mg 1 mg/ml -Cellstain- PI solution 668.39 1 ml 1 mg/ml! UV -Cellstain- AO 301.81 1 mg 1 mg/ml -Cellstain- AO solution 301.81 1 ml 1 mg/ml 1-10 g/ml ( PBS(-) -Cellstain- Hoechst 33258 solution 533.88 1 ml 1 mg/ml ) -Cellstain- Hoechst 33342 solution 561.93 1 ml 1 mg/ml! UV DMSO (DMSO ) -Cellstain- MitoRed 637.17 50 μg 8 1 mmol/l 20-200 nmol/l -Cellstain- Rh123 380.82 1 mg 1 mg/ml 20-100 nmol/l!
3 1. 2. 3. 10 4 10 5 cells 24 24 PBS(-)1 ml 30 50% PBS(-) 0.2 ml 37 15 30 PBS(-)0.5 ml
1,000 rpm, 3 PBS(-) 10 5 10 6 cells/ml 30 µl 15 µl 37 15 30 10 µl
10 4 10 5 cells 24 24 PBS(-)1 ml 30 50% PBS(-) 70%EtOH 1.0 ml 70-100% EtOH 100% MeOH -20-20 15 70%EtOH PBS(-)1 ml PBS(-) 0.2 ml 37 15 30 PBS(-)0.5 ml
HeLa 1. BCECF-AM (x200, B ) Calcein-AM (x400, B ) CFSE (x400, B ) CytoRed (x200, G ) FDA (x400, B ) MitoRed (x200, G ) Rh123 (x200, B ) AO (x200, B ) Hoechst 33258 (x200, U )
HeLa 3. EB (x300, G ) DAPI (x200, U ) PI (x 200, G ) Cell Number 3% NIH3T3 Hoechst 33258 HyLite Fluor TM 555 Fluorescent Intensity HL60 Calcein-AM 488nm ( ( ) 0.5%Triton X-100 3 4% 0.5%Triton X-100
- Cellstain - Double Staining Kit ( ) Calcein-AM PI (Propidium Iodide) - Cellstain - Double Staining Kit CS01 A : Calcein-AM stock solution (1 mmol/l) B : PI stock solution (1.5 mmol/l) 4 vials 1 vial! -20 A Calcein-AM PBS(-) A B 5 ml PBS(-) A 10 l B 15 l! Calcein-AM: 2 mol/l, PI: 4 mol/l PI UV
HeLa 1,000 rpm, 3 PBS(-) 10 5 10 6 cells/ml 200 l 100 l 37 15 30 10 l
490±10 nm 545 nm Calcein-AM PI 0.1 10 mol/l PI 0.1% 0.1 0.5% 10 70% 30 0.1 10 mol/l Calcein-AM Calcein-AM -Cellstain- Double Staining Kit MHD-1 (480 nm [ ]
PBS (Pluronic F-127) Probenecid) -Cellstain- CFSE 1mg
DMSO DMSO DMSO CFSE 8 Calcein-AMBCECF-AM 3 ES. A. Weston, et al., J. Immunol.Methods, 1990, 133, 87. EH. P. Zhong, et al., Hum. Immunol., 1993, 37, 264. Calcein-AMBCECF-AM EL. S. D. Clerck, et al., J. Immunol. Methods, 1994, 172, 115. BCECF-AM ph Calcein-AM C F S E C y t o R e d Calcein-AM FDA Calcein-AMBCECF-AMCFDACFSE L. S. D. Clerck, et al., J. Immunol. Methods, 1994. 172, 115. Calcein-AM BCECF-AM AO PIEBDAPI FDA Appl. Microbiol. Biotechnol., 1992, 38, 268.
A O 2 2 Hoechst 33258Hoechst 33342 DNA - DNA Hoechst33342 E B DNARNA P I EB EB DAPI2 (minor groove) - MitoRed Rh123 MitoRedRh123 Rhodamine Rhodamine
-Cellstain- Double Staining Kit Calcein-AM PI Calcein-AM Calcein (AM) Calcein- AM AM PI DNA PI 490 10 nm 545 nm Calcein-AM Calcein-AM PI 33 Calcein-AM L. S. D. Clerck, et al., J. Immunol. Methods, 1994. 172, 115. -20Calcein-AM PI -20 1 PI UV
(12) (11) (28) (4) (2) (1) J. Immunoi.