DOJINプロトコル修正.indd
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- あきお そめや
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2 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 solution -Cellstain- Hoechst solution -Cellstain- MitoRed -Cellstain- Rh123 -Cellstain- Double Staining Kit p. 37
3 3 H MTT WST 3 H RI DNA RI MTT WST 19
4 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 : nm Cell Counting Kit-8 : nm Cell Counting Kit CK Cell Counting Kit -8CK04 Cell Counting Kit A B!! Cell Counting Kit-8!!
5 Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit Cell Counting Kit-8 Cell Counting Kit Cell Counting Kit-8 cells /ml Cell Counting Kit Cell Counting Kit /10 Cell Counting Kit : nm Cell Counting Kit-8 : nm HeLa HL60
6 Cell Counting Kit-8 Cell Counting Kit HeLa HL , , ,0 cells well Cell Counting Kit cells/ml HeLa HL HeLa 0.5 HL Absorbance Absorbance 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 [ 3 H]-Thymidine x 10-5 dpm
7 Cell Counting Kit-8 Cell Counting Kit cells /ml
8 Cell Counting Kit-8 Cell Counting Kit 96 Cell Counting Kit 96 Cell Counting Kit / 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(-)
9 Cell Counting Kit-8 Cell Counting Kit w /v 10
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) Cell Counting Kit (Cell Counting Kit-8) Cell Counting Kit (Cell Counting Kit-8)
11 Cell Counting Kit-8 Cell Counting Kit 24 well 12 well 96 well ml well 1 w/v% SDS mol/ l (10 ) Cell Counting Kit (Cell Counting Kit-8) nm nm nm nm nm 1 8
12 Cell Line Description Reference 293T human kidney carcinoma H. Fuda, et al., J. Lipid Res., 2007, 48, T3-L1 mouse embryonic fibroblast D. Huang, et al., FASEB J., 2005, 19, A431 human epithelial carcinoma cell H. Ohori, et al., Mol. Cancer Ther., 2006, 5, A549 human lung cancer cell C. A. Reilly, et al., Toxicol. Sci., 2003, 73, 170. ; T. Kitamuro, et al., J. Biol. Chem., 2003, 278, J. Stankova, et al., Clin. Cancer Res., 2005, 11, ;I.Imoto, et al., Cancer Res., 2006, 66, ; S. Semba, et al., J. Biol. Chem., 2006, 281, Alexander cell pancreatic cancer cell S. Awale, et al., Cancer Res., 2006, 66, 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, AsPC-1 pancreatic cancer cell T. Mori, et al., Mol. Cancer Ther., 2004, 3, 29. ; S. Awale, et al., Cancer Res., 2006, 66, B16F1 murine malignant melanoma S. Shibata, et al., J. Immunol., 2006, 177, BBMVEC bovine brain microvascular endothelial T. Kitamuro, et al., J. Biol. Chem., 2003, 278, 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, C33A human cervical carcinoma W. Yang, et al., Mol. Cancer Ther., 2006, 5, 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, mouse M. Ikonen, et al., Proc. Natl. Acad. Sci. USA, 2003, 100, T. Kitamuro, et al., J. Biol. Chem., 2003, 278, ; H. Bando, et al., Clin. Cancer Res., 2005, 11, African green monkey kidney cell CV-1 African green monkey kidney cell J. Peloponese, et al., J. Biol. Chem., 2006, 281, Daoy human medulloblastoma X. Li, et al., Mol. Cancer Ther., 2005, 4, 1912.; S. Kim, et al., Clin. Cancer Res., 2006, 12, 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, DT40 chicken B-lymphocyte cell F. Shinozaki, et al., J. Biol. Chem., 2006, 281, DU145 human prostate carcinoma P. Davis-Searles, et al., Cancer Res., 2005, 65, ; D. J. Son, et al., Mol. Cancer Ther., 2007, 6, 675. G361 human melanoma cell H. Ohori, et al., Mol. Cancer Ther., 2006, 5, GCIY human gastric carcinoma H. Ohori, et al., Mol. Cancer Ther., 2006, 5, H441 human pulmonary adenocarcinoma H. Shimura, et al., Cancer res., 2001, 61, 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, H. Shimura, et al., Cancer res., 2001, 61, 3640.; C. Shi, et al., J. Biol. Chem., 2004, 279, 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, ; H. Ohori, et al., Mol. Cancer Ther., 2006, 5, from Wistar rat embryos K. Kurata, et al., J. Pharmacol. Exp. Ther., 2004, 311, 237. D. Huang, et al., FASEB J., 2005, 19, ;L. Song, et al., Stem Cells, 2006, 24, human mesenchymal stem cell ILT-Hod T-cell T. Kasai, et al., J. Biol. Chem., 2002, 277, Jurkat human T cell Kasumi-1 acute myeloid leukemia cell G. Zhou, et al., Blood, 2007, 109, 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, ; L. Lu, et al., J. Biochem., 2007, 141, 157. I. Imoto, et al., Cancer Res., 2001, 61, ;K. Nakakuki, et al., Carcinogenesis, 2002, 23, 19. L929 mouse fibroblast Y. Morita, et al., Proc. Natl. Acad. Sci. USA, 2000, 97, LK87 human lung adenocarcinoma H. Ohori, et al., Mol. Cancer Ther., 2006, 5, 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, MDCK canine kidney epithelial cell H. Shimura, et al., Cancer res., 2001, 61, 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, 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, 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, PCN primary cortical neuron T. Chiba, et al., J. Neurosci., 2005, 25, PSN-1 pancreatic cancer cell S. Awale, et al., Cancer Res., 2006, 66, 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, SH-SY5Y human neuroblastoma T. Nakagawa, et al., Mol. Cell. Biol., 2002, 22, SK-N-SH human neuroblastoma Y. Wang, et al., J. Virol., 2004, 78, 7916.
13 MTT MTT Cell Counting Kit [ 3 H] MTT DMSO 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
14 MTT MTT MTT cells /ml MTT PBS(-) /10 DMSO DMSO535
15 MTT HeLa , , ,0 cellswell MTT cells/ml 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
16 MTT cells /ml l MTT 10 1/10
17 MTT 2 4 PBS() MTT 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
18 MTT 650 nm MTT MTT MTT 2 4 MTT DMSO MTT MTT
19 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
20 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 nm nm Cell Counting Kit -F06 PBS(-)Ca 2+, Mg 2+! Cell Counting Kit-F Cell Counting Kit-F PBS(-) 50!
21 Cell Counting Kit-F Cell Counting Kit-F Cell Counting Kit-F cells/ml PBS(-) 100 V 96 Cell Counting Kit-F 96 1/ nm nm
22 Cell Counting Kit-F HeLa HL , , ,0 cellswell Cell Counting Kit-F cells/ml HeLa HL60 Cell Counting Kit-F HeLa HL60 Relative Fluorescence Intensity Intensity Relative Fluorescence Intensity Intensity Number of Cells (x10 3 cells/well) Number of Cells Number of Cells (x10 3 cells/well) Number of Cells
23 Cell Counting Kit-F cells /ml
24 Cell Counting Kit-F PBS(-) Cell Counting Kit-F / nm 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
25 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
26 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
27 MTT WST-1, WST-8, XTT, MTS NADH NADH ATP ATP ATP 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
28 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
29 Calcein-AMFluorescein diacetate (FDA)CFSE fluorescein Propidium iodide (PI)Ethidium bromide (EB) DAPI DNA
30 -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 Cellstain- Hoechst Cellstain- MitoRed -Cellstain- Rh123 ( DMSO (DMSO ) -Cellstain- Calcein-AM mg 0.5-1mmol/l -Cellstain- Calcein-AM solution ml 1mmol/l 1-20 mol/l -Cellstain- CFSE mg 0.5-1mmol/l ( PBS(-) -Cellstain- CytoRed solution ml 1mmol/l ) -Cellstain- FDA mg 0.5-1mmol/l BCECF-AM Special packging µg x mmol/l! PBS(-)
31 -Cellstain- DAPI mg 1 mg/ml -Cellstain- DAPI solution ml 1 mg/ml of Buffer μ -Cellstain- EB mg 1 mg/ml -Cellstain- EB solution ml 1 mg/ml -Cellstain- PI mg 1 mg/ml -Cellstain- PI solution ml 1 mg/ml! UV -Cellstain- AO mg 1 mg/ml -Cellstain- AO solution ml 1 mg/ml 1-10 g/ml ( PBS(-) -Cellstain- Hoechst solution ml 1 mg/ml ) -Cellstain- Hoechst solution ml 1 mg/ml! UV DMSO (DMSO ) -Cellstain- MitoRed μg 8 1 mmol/l nmol/l -Cellstain- Rh mg 1 mg/ml nmol/l!
32
33 cells PBS(-)1 ml 30 50% PBS(-) 0.2 ml PBS(-)0.5 ml
34 1,000 rpm, 3 PBS(-) cells/ml 30 µl 15 µl µl
35 cells PBS(-)1 ml 30 50% PBS(-) 70%EtOH 1.0 ml % EtOH 100% MeOH %EtOH PBS(-)1 ml PBS(-) 0.2 ml PBS(-)0.5 ml
36 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 (x200, U )
37 HeLa 3. EB (x300, G ) DAPI (x200, U ) PI (x 200, G ) Cell Number 3% NIH3T3 Hoechst HyLite Fluor TM 555 Fluorescent Intensity HL60 Calcein-AM 488nm ( ( ) 0.5%Triton X % 0.5%Triton X-100
38 - 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
39 HeLa 1,000 rpm, 3 PBS(-) cells/ml 200 l 100 l l
40 490±10 nm 545 nm Calcein-AM PI mol/l PI 0.1% % 10 70% mol/l Calcein-AM Calcein-AM -Cellstain- Double Staining Kit MHD-1 (480 nm [ ]
41 PBS (Pluronic F-127) Probenecid) -Cellstain- CFSE 1mg
42 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, , 115. Calcein-AM BCECF-AM AO PIEBDAPI FDA Appl. Microbiol. Biotechnol., 1992, 38, 268.
43 A O 2 2 Hoechst 33258Hoechst DNA - DNA Hoechst33342 E B DNARNA P I EB EB DAPI2 (minor groove) - MitoRed Rh123 MitoRedRh123 Rhodamine Rhodamine
44 -Cellstain- Double Staining Kit Calcein-AM PI Calcein-AM Calcein (AM) Calcein- AM AM PI DNA PI nm 545 nm Calcein-AM Calcein-AM PI 33 Calcein-AM L. S. D. Clerck, et al., J. Immunol. Methods, , Calcein-AM PI PI UV
45 (12) (11) (28) (4) (2) (1) J. Immunoi.
46
細胞増殖測定細胞染色プロトコル
Cell Counting Kit Cell Counting Kit -8 3 Cell Counting Kit -F -Cellstain- -Cellstain- -Cellstain- Calcein-AM solution -Cellstain- CytoRed solution -Cellstain- BCECF-AM special packaging -Cellstain- -Cellstain-
Various function and toxicity of iron Key words 1) Hentze, M. W. et al: Cell, 117, 285-297 (2004) 2) Ganz, T.: Blood, 102, 783-788 (2003) 3) Nemeth, E. et al.: Science, 306, 2090-2093 (2004) 4) Hentze,
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1) van Meer, G., Op den Kamp, J. A.: J. Cell. Biochem., 19, 193-2040982) 2) Gaffet, P., Bettache, N., Bienvenue, A.: Biochemistry, 34, 6762-6769(1995) 3) Sims, P. J., Wiedmer, T.: Thromb. Haemost., 86,
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1) Asa, S. L., et al.: Immunohistochemical localization of neuron-specific enolase in the human hypophysis and pituitary adenoma, Arch. Pathol. Lab. Med., 108: 40 `43, 1984. 2) Chi, B. H. and Kim, R. C.:
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CODE No. 323 For Research Use Only, Not for use in diagnostic procedures. Ab-Match Assembly Human PAP1 (REG3α) Kit For technical material or related information, please refer to http://ruo.mbl.co.jp/product/abmatch/abmatch.html.
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