-SulfoBiotics- SSP4 Technical Manual ࡣࡌࡵ ㏆ᖺࠊ◲㯤ཎᏊࡀ㐃⤖ࡋࡓࣃ࣮ࢫࣝࣇࢻࡸ࣏ࣜࢫࣝࣇࢻࡢࡼ࠺࡞ࢧࣝࣇ࢙ࣥ◲㯤 (Sulfane Sulfur) ࢆྵࡴศᏊ ✀ࡀ⏕యෆከࡃᏑᅾࡋ࡚࠸ࡿࡇࡀ᫂ࡽࡉࢀ࡚࠸ࡲࡍࠋࡇࡢࡼ࠺࡞ศᏊ✀ࡣࠊ◲Ỉ⣲ࡢ⏘⏕ࡸ㈓ⶶࠊᨺฟ ࡔࡅ࡛࡞ࡃࠊS- ࢫࣝࣇࣄࢻࣜࣝ࡞ࡢࢱࣥࣃࢡ㉁ෆࢳ࣮࢜ࣝࢆࢱ࣮ࢤࢵࢺࡋࡓࢩࢢࢼࣝఏ㐩ࡶ㛵ࡋ࡚࠸ ࡿࡇࡀ♧၀ࡉࢀ࡚࠾ࡾࠊ㠀ᖖὀ┠ࡉࢀ࡚࠸ࡲࡍࠋSSP4 (Sulfane Sulfur Probe 4) ࡣࠊࢧࣝࣇ࢙ࣥ◲㯤≉␗ⓗ ᛂࡋ࡚ᙉ࠸⺯ග (λex=482 nm, λem=515 nm) ࢆⓎࡍࡿヨ⸆࡛࠶ࡾࠊࢧࣝࣇ࢙ࣥ◲㯤ྵ᭷ศᏊ✀ࡢ⺯ග᳨ฟࡸ⣽ ⬊ෆゎᯒ᭷⏝࡛ࡍࠋ ͤᮏヨ⸆ࢆ⣽⬊ෆࢧࣝࣇ࢙ࣥ◲㯤ྵ᭷ศᏊࡢゎᯒ⏝ࡍࡿሙྜࠊヨ⸆ࢆ⣽⬊ෆᑟධࡍࡿࡓࡵ࢝ࢳ࢜ࣥᛶ⏺㠃άᛶ࡛࠶ࡿ Cetyltrimethylammonium bromide (CTAB) ࢆ⏝ࡋ࡚ࡃࡔࡉ࠸ࠋ Fig. 1 SSP4 ࢧࣝࣇ࢙ࣥ◲㯤ࡢᛂ Fig. 2 ࢧࣝࣇ࢙ࣥ◲㯤ࡢᛂక࠺ SSP4 ࡢບ㉳࠾ࡼࡧ⺯ගࢫ࣌ࢡࢺࣝኚ ෆᐜ ಖᏑ᮲௳ ᚲせ࡞ࡶࡢ ⁐ᾮㄪ〇 Fig. 3 ࢧࣝࣇ࢙ࣥ◲㯤 (Sulfane Sulfur) ࢆྵࡴศᏊ✀ SSP4 1 mg x 1 㐽ගࠊ-20Υ௨ୗ࡛ಖᏑࡋ࡚ࡃࡔࡉ࠸ࠋ - Dimethyl sulfoxide (DMSO) - ↓⾑Ύᇵᆅ - PBS - Cetyltrimethylammonium bromide (CTAB) - ࣐ࢡࣟࣆ࣌ࢵࢺ - 10 mmol/l SSP 4 Stock Solution SSP4 1mg ࢆྵࡴࢳ࣮ࣗࣈ DMSO 165 µl ࢆῧຍࡋࠊࣆ࣌ࢵࢸࣥࢢࡼࡾ⁐ゎࡍࡿࠋ ͤࡇࡢ⁐ᾮࢆᑡ㔞ᑠศࡅࡋࠊ-20 Υ௨ୗ࡛ಖᏑࡋ࡚ࡃࡔࡉ࠸ࠋ⣙ 2 ࣨ᭶㛫ࡣᏳᐃ࡛ࡍࠋ - 0.5 mmol/l CTAB ࢆྵࡴ↓⾑Ύᇵᆅ CTAB 36.4mg ࢆ 1.5ml 㐲ᚰࢳ࣮ࣗࣈ⛗㔞ࡋࠊ㉸⣧Ỉ 1 ml ࢆῧຍࡍࡿࠋࢻ࣮ࣛࣖ࡞࡛ຍ ⁐ゎࡋࠊ100 mmol/l CTAB Ỉ⁐ᾮࡍࡿࠋࡇࡢỈ⁐ᾮࢆ↓⾑Ύᇵᆅ࡛ 200 ಸᕼ㔘ࡋࠊ0.5 mmol/l CTAB ࢆྵࡴ↓⾑Ύᇵᆅࡍࡿࠋ - SSP4 Working Solution ⤊⃰ᗘࡀ 5-20 µmol/l ࡞ࡿࡼ࠺ 10 mmol/l SSP4 Stock Solution ࢆࠊ0.5 mmol/lCTAB ࢆྵࡴ↓⾑Ύᇵᆅῧ ຍࡍࡿࠋ 㸸10 mmol/l SSP4 Stock Solution 10 µl ࢆ 0.5 mmol/l CTAB ࢆྵࡴ↓⾑Ύᇵᆅ 5 ml ῧຍࡍࡿࠊ20 µmol/l SSP4 Working Solution ࡞ࡿࠋ ᧯స 1) ⺯ග㢧ᚤ㙾ほᐹࡀྍ⬟࡞ࣉ࣮ࣞࢺ࠶ࡿ࠸ࡣࢹࢵࢩࣗ⣽⬊ࢆ✀ࡋࠊᇵ㣴ࡍࡿࠋ 2) ୖΎࢆ㝖ཤࡋࡓᚋࠊ⣽⬊ࢆ↓⾑Ύᇵᆅ࡛Ὑίࡍࡿࠋ 3) ㄪ〇ࡋࡓ SSP4 Working Solution ࢆ⣽⬊ῧຍࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮ (37Υ ) ෆ࡛ 15 ศ㛫㟼⨨ࡍࡿࠋ 4) ୖΎࢆ㝖ཤࡋࡓᚋࠊPBS ࡛ 2 ᅇὙίࡍࡿࠋ 5) PBS ࢆῧຍࡋࠊ⺯ග㢧ᚤ㙾࡛ほᐹࡍࡿࠋ SB10: -SulfoBiottics- SSP4 Revised February 06, 2015 ᐇ㦂 1 ࢧࣝࣇ࢙ࣥ◲㯤ࢻࢼ࣮࠾ࡼࡧࢧࣝࣇ࢙ࣥ◲㯤ᾘཤῧຍࡼࡿ⣽⬊ෆࢧࣝࣇ࢙ࣥ◲㯤㔞ኚࡢゎᯒ 1) 96-well black clear bottom plate CHO ⣽⬊ ࢆ⾑Ύධࡾᇵᆅ㸦DMEM104 cells/well ࡞ࡿࡼ࠺✀ࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮㸦37Υ㸧ෆ୍࡛ᬌᇵ㣴ࡋࡓࠋ 2) ୖΎࢆ㝖ཤࡋࡓᚋࠊ↓⾑Ύᇵᆅ㸦DMEM㸧࡛⣽⬊ࢆ 2 ᅇὙίࡋࡓࠋ 3) 100 µmol/l ࢧࣝࣇ࢙ࣥ◲㯤ࢻࢼ࣮ Na2S3 (Sodium trisulfide) ࠊ࠶ࡿ࠸ࡣ 1 mmol/l ࢧࣝࣇ࢙ࣥ◲㯤ᾘཤ NaCN (Sodium cyanide) ࢆྵࡴ↓⾑Ύᇵᆅ㸦DMEM㸧100 µl ࢆྛ࢙࢘ࣝῧຍࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮㸦37Υ㸧ෆ ࡛ 15 ศ㛫㟼⨨ࡋࡓࠋ 4) ୖΎࢆ㝖ཤࡋࡓᚋࠊ↓⾑Ύᇵᆅ㸦DMEM㸧࡛⣽⬊ࢆ 2 ᅇὙίࡋࡓࠋ 5) 20 µmol/l SSP4 Working Solution 100 µl ࢆ࢙࢘ࣝῧຍࡋࠊCO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮㸦37Υ㸧ෆ࡛ 15 ศ㛫㟼⨨ࡋࡓࠋ 6) ୖΎࢆ㝖ཤࡋࡓᚋࠊPBS ࡛⣽⬊ࢆ 2 ᅇὙίࡋࡓࠋ 7) PBS 100 µl ࢆ࢙࢘ࣝῧຍࡋࡓᚋࠊ⺯ග㢧ᚤ㙾ࢆ⏝࠸࡚⣽⬊ࢆほᐹࡋࡓࠋ ᮍฎ⌮ࠉࠉࠉࠉࠉࠉࠉࠉࠉࠉࠉ100 µmol/l Na2S3 ฎ⌮ࠉ ࠉ 㸦㟢ග㛫 1000 msec㸧 Na2S3㸦ࢧࣝࣇ࢙ࣥ◲㯤ࢻࢼ࣮㸧࡛ฎ⌮ࡋࡓ⣽⬊ෆᙉ࠸⺯ගࡀほᐹࡉࢀࡓࠋ 1 mmol/l NaCN ฎ⌮ ᮍฎ⌮ 㸦㟢ග㛫 2000 msec㸧 NaCN㸦ࢧࣝࣇ࢙ࣥ◲㯤ᾘཤ㸧࡛ฎ⌮ࡋࡓ⣽⬊ෆࡣࠊ ᮍฎ⌮ࡢ⣽⬊㸦ෆᅉᛶࡢࢧࣝࣇ࢙ ࣥ◲㯤㸧࡛ほᐹࡉࢀࡓ⺯ගࡀほᐹࡉࢀ࡞ࡗࡓࠋ ᐇ㦂 2 - ◲Ỉ⣲㸦H2S㸧άᛶ㓟⣲㸦❅⣲㸧✀ࡢᛂࡼࡿࢧࣝࣇ࢙ࣥ◲㯤ྵ᭷≀㉁ࡢ⏕ᡂ☜ㄆ 1) 100 µmol/l άᛶ㓟⣲㸦❅⣲㸧✀ 100 µmol/l Na2S ࢆྵࡴ⁐ᾮ (PBS) ࢆᐊ ࡛ 10 ศ㛫㟼⨨ࡋࡓࠋ 2) ᧯స 1) ࡢ⁐ᾮࠊ⤊⃰ᗘࡀ 10 µmol/l ࡞ࡿࡼ࠺ 10 mmol/l SSP4 Stock Solution ࢆῧຍࡋࠊᐊ ࡛ 30 ศ㛫㟼 ⨨ࡋࡓࠋ 3) ᧯స 2) ࡢ⁐ᾮࡢ 515 nm ⺯ගᙉᗘ (λex=482 nm) ࢆࠊ⺯ගศග㧗ᗘィࢆ⏝࠸࡚ ᐃࡋࡓࠋ ◲Ỉ⣲㸦H2S㸧ྛάᛶ㓟⣲㸦❅⣲㸧✀ࡢᛂࡼࡗ࡚ࢧࣝࣇ࢙ࣥ◲㯤ྵ᭷ศᏊࡀ⏕ᡂࡉࢀࡿࡇࡀ☜ㄆࡉࢀࡓࠋ ࡲࡓࠊࡑࡢ୰࡛ࡶ◲Ỉ⣲㸦H2S㸧୍㓟❅⣲㸦NO㸧ࡢᛂࡣ㠀ᖖຠ⋡ࡀ㧗࠸ࡇࡀ♧ࡉࢀࡓࠋ ᮏヨ⸆ࡣࠊࢧࣝࣇ࢙ࣥ◲㯤ྵ᭷ศᏊࡢ⏕ᡂゎᯒࡶ᭷⏝࡛࠶ࡿࠋ ཧ⪃ᩥ⊩ 1) W. Chen, C. Liu, B. Peng, Y. Zhao, A. Pacheco and M. Xian, Chem. Sci., 2013, 4, 2892. 2) T. Ida, T. Sawa, H. Ihara. Y. Tsuchiya, Y. Watanabe, Y. Kumagai, M. Suematsu, H. Motohashi, S. Fujii, T. Matsunaga, M. Yamamoto, K. Ono, N. O. Devarie-Baez, M. Xian, J. M Fukuto, and T. Akaike, Proc. Natl. Acad. Sci. USA., 2014, 111, 7606. 3) E. Marutani, M. Sakaguchi, W. Chen, K. Sasakura, J. Liu, M. Xian, K. Hanaoka, T. Nagano, and F. Ichinose, Med. Chem. Commun., 2014, 5, 1577. 4) M. Sakaguchi, E. Marutani, H-S. Shin, W. Chen, K. Hanaoka, M. Xian, and F. Ichinose, Anesthesiology. 2014, 121, 1248. ࡈ㉁ၥ࣭ࡈせᮃࡣୗグࡲ࡛࠾ၥ࠸ྜࢃࡏࡃࡔࡉ࠸ࠋ 㸺㛤Ⓨඖ㸼 Dojindo Molecular Technologies, Inc. 30W Gude Dr, Suite 260, Rockville, Maryland, 20850 U.S.A. Tel: +1-301-987-2667, Fax: +1-301-987-2687 URL: www.dojindo.com 㸺ጤク〇㐀ඖ㸼 ᰴᘧ♫ ྠோᏛ◊✲ᡤ ⇃ᮏ┴ୖ┈ᇛ㒆┈ᇛ⏫⏣ཎ 2025-5 ࠛ 861-2202 Tel:096-286-1515 Fax:096-286-1525 URL:www.dojindo.co.jp/ ࢻ࣮ࢪ࣭࣮ࣥࢫࢺ ( ᮾி ) Tel:03-3578-9651 Fax:03-3578-9650 SB10: -SulfoBiottics- SSP4
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