{"id":3399,"date":"2013-04-10T11:38:09","date_gmt":"2013-04-10T02:38:09","guid":{"rendered":"http:\/\/cell.brc.riken.jp:7321\/?page_id=3399"},"modified":"2016-09-01T13:47:57","modified_gmt":"2016-09-01T04:47:57","slug":"bioassay_l929","status":"publish","type":"page","link":"http:\/\/cell.brc.riken.jp\/ja\/rcb\/bioassay_l929","title":{"rendered":"L929(RCB1422,RCB1451,RCB2619)\u3092\u7528\u3044\u305fTNF-\u03b1\u611f\u53d7\u6027\u8a66\u9a13\u306b\u3064\u3044\u3066"},"content":{"rendered":"<p>L929\u304cTNF\u03b1\u306b\u3088\u3063\u3066\u969c\u5bb3\u3092\u53d7\u3051\u308b\u6027\u8cea\u3092\u5229\u7528\u3057\u3066\u3001TNF\u03b1\u611f\u53d7\u6027\u8a66\u9a13\u3092\u884c\u3046\u65b9\u6cd5\u3067\u3059\u3002\u4fdd\u6709\u3057\u3066\u3044\u308b3\u682a\u306eL929\u7d30\u80de\uff08RCB1422\u3001RCB1451\u3001RCB2619\uff09\u3092\u4f7f\u7528\u3057\u3001\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u3092\u7528\u3044\u3066\u611f\u53d7\u6027\u306e\u9055\u3044\u3092\u6bd4\u8f03\u3059\u308b\u65b9\u6cd5\u3092\u3054\u7d39\u4ecb\u3057\u307e\u3059\u3002<\/p>\n<p>*\u30d0\u30a4\u30aa\u30a2\u30c3\u30bb\u30a4\u306b\u3064\u3044\u3066<br \/>\n\u3053\u306e\u65b9\u6cd5\u3067\u65e2\u77e5\u306eTNF\u03b1\u3092\u7528\u3044\u3066\u691c\u91cf\u7dda\uff08\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u3067\u67d3\u8272\u3055\u308c\u305f\u5272\u5408\uff09\u3092\u4f5c\u6210\u3057\u305f\u5f8c\u3001\u691c\u4f53\u306e\u5024\u3068\u691c\u91cf\u7dda\u3092\u6bd4\u3079\u308b\u3053\u3068\u306b\u3088\u308a\u3001\u691c\u4f53\u306eTNF\u03b1\u6fc3\u5ea6\u3092\u6e2c\u5b9a\u3059\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002<\/p>\n<p><\u53c2\u8003\u6587\u732e><br \/>\nCurrent Protocols in Immunology. 2001 Chapter 6 : Unit 6.10\u3000\u300cMeasurement of TNF\u03b1 and TNF\u03b2\u300d<\/p>\n<p><strong>1. \u6e96\u5099\u3059\u308b\u3082\u306e<\/strong><\/p>\n<p>(1)\u7d30\u80de\u3068\u57f9\u5730<\/p>\n<ul>\n<li>L929\u7d30\u80de\uff08RCB1422, RCB1451, RCB2619\uff09\n<li>\u57f9\u990a\u57f9\u5730<br \/>\n\u3000\u3000RCB1422 : MEM+10%HS+0.1mM NEAA<br \/>\n\u3000\u3000RCB1451 : MEM+5%CS<br \/>\n\u3000\u3000RCB2619 : RPMI1640+10%FBS\n<\/ul>\n<p>\u3000\u203b\u4eca\u56de\u306f3\u7d30\u80de\u306e\u6bd4\u8f03\u3092\u884c\u3046\u305f\u3081\u3001\u30a2\u30c3\u30bb\u30a4\u306e\u6642\u306e\u307f\u300cMEM+10%HS+0.1mM NEAA\u300d\u3092\u4f7f\u7528\u3002<\/p>\n<p>(2)\u8a66\u85ac<\/p>\n<ul>\n<li>BSA\uff08\u5ca9\u4e95\u5316\u5b66\u85ac\u54c1\u793e, #0125R\uff09<br \/>0.1% BSA\u6eb6\u6db2\u30920.22\u03bcm\u30d5\u30a3\u30eb\u30bf\u30fc\u3067\u6ffe\u904e\u6ec5\u83cc\u3059\u308b\u3002\n<li>TNF\u03b1\uff08SIGMA\u793e, #T7539\uff09<br \/>\u6ffe\u904e\u6ec5\u83cc\u5f8c\u306e0.1% BSA\u6eb6\u6db21ml\u306b10\u03bcg\u306eTNF\u03b1\u3092\u6eb6\u89e3\u3059\u308b\u3002\uff0810\u03bcg\/ml sol.\uff09\n<li>Actinomycin D\uff08SIGMA\u793e, #A9415\uff09<br \/>\u30b9\u30c8\u30c3\u30af\u6eb6\u6db2\u306f\u3001500\u03bcg\/ml\u3068\u306a\u308b\u3088\u3046\u306b\u30e1\u30bf\u30ce\u30fc\u30eb\u306b\u6eb6\u89e3\u3057\u30010.22\u03bcm\u30d5\u30a3\u30eb\u30bf\u30fc\u3067\u6ffe\u904e\u6ec5\u83cc\u3059\u308b\u3002<br \/>\u4f7f\u7528\u6642\u306b4\u03bcg\/ml\u3068\u306a\u308b\u3088\u3046\u306b\u57f9\u5730\u3067\u5e0c\u91c8\u3059\u308b\u3002<br \/>\uff08\u6ce8:Actinomycin D \u306f\u3001\u7528\u6642\u89e3\u51cd\u3067\u4f7f\u7528\u3059\u308b\u3002\u51cd\u7d50\u878d\u89e3\u306f\u7e70\u308a\u8fd4\u3055\u305a\u3001\u4f7f\u3044\u304d\u308a\u304c\u671b\u307e\u3057\u3044\u3002)\n<li>10% \u4e2d\u6027\u7de9\u885d\u30db\u30eb\u30de\u30ea\u30f3\u6eb6\u6db2\uff08\u548c\u5149\u7d14\u85ac\u793e, #062-01661\uff09\n<li>0.4% \u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u6eb6\u6db2\uff08\u6771\u4eac\u5316\u6210\u5de5\u696d\u793e, #C0428\uff09\n<li>PBS(+)\n<li>\u30e1\u30bf\u30ce\u30fc\u30eb\uff08SIGMA\u793e, #19-2410\uff09<br \/>\n\u6e2c\u5b9a\u5f53\u65e5\u306b70%\u6eb6\u6db2\u3068\u306a\u308b\u3088\u3046\u306b\u5e0c\u91c8\u3059\u308b\u3002\u767a\u71b1\u3059\u308b\u306e\u3067\u5ba4\u6e29\u307e\u3067\u51b7\u307e\u3057\u3066\u304b\u3089\u4f7f\u7528\u3059\u308b\u3002\n<\/ul>\n<p>(3)\u5bb9\u5668<\/p>\n<ul>\n<li>96\u30a6\u30a7\u30eb\u3000\u30de\u30a4\u30af\u30ed\u30d7\u30ec\u30fc\u30c8\uff08\u5e73\u5e95\uff09\uff08BD Falcon\u793e, #353072\uff09\n<\/ul>\n<p><strong>2.\u65b9\u6cd5<\/strong><br \/>\n(1)\u7d30\u80de\u306e\u64ad\u7a2e<\/p>\n<ol class=\"list_parentheses\">\n<li>96\u30a6\u30a7\u30eb\u3000\u30de\u30a4\u30af\u30ed\u30d7\u30ec\u30fc\u30c8\u306bL929\u7d30\u80de\u30922&#215;10<sup>4<\/sup> cells\/100\u03bcl\/well\u3067\u64ad\u7a2e\u3059\u308b\u3002\n<li>37\u2103, CO<sub>2<\/sub> 5%\u306e\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30bf\u30fc\u3067\u4e00\u6669\u57f9\u990a\u3059\u308b\u3002\n<\/ol>\n<p>(2)TNF\u03b1\u306e\u6dfb\u52a0<\/p>\n<ol class=\"list_parentheses\">\n<li>\u5225\u5bb9\u5668\u3067TNF\u03b1\u3092\u5e0c\u91c8\u3059\u308b\u3002\uff08\u5b9f\u9a13\u3067\u306f\u6bb5\u968e\u5e0c\u91c8\u306b\u3088\u308a0.01\uff5e10ng\/ml\u306b\u8abf\u88fd\u3057\u305f\u3082\u306e\u3068\u3001100ng\/ml\u306b\u8abf\u88fd\u3057\u305f\u3082\u306e\u3092\u4f7f\u7528\uff09\n<li>\u64ad\u7a2e\u7fcc\u65e5\u306b\u7d30\u80de\u304c\u30b3\u30f3\u30d5\u30eb\u30a8\u30f3\u30c8\u306b\u306a\u3063\u3066\u3044\u308b\u3053\u3068\u3092\u78ba\u8a8d\u3057\u3001\u57f9\u5730\u3092\u9664\u304f\u3002\uff08\u6ce8\uff1aL929\u306f\u5fc5\u305a\u30b3\u30f3\u30d5\u30eb\u30a8\u30f3\u30c8\u306e\u72b6\u614b\u3067\u7528\u3044\u308b\u3053\u3068\u3002\u30b3\u30f3\u30d5\u30eb\u30a8\u30f3\u30c8\u306b\u306a\u3089\u306a\u3044\u5834\u5408\u306f\u3001\u64ad\u7a2e\u7d30\u80de\u6570\u3092\u5897\u3084\u3059\u304b\u3001\u57f9\u990a\u6642\u9593\u3092\u9577\u304f\u3059\u308b\u3002\uff09\n<li>\u65b0\u305f\u306b\u57f9\u5730\u3092100\u03bcl\/well\u3067\u6dfb\u52a0\u3059\u308b\u3002\n<li>1)\u3067\u4f5c\u6210\u3057\u305fTNF\u03b1\u6eb6\u6db2\u3092\u30013)\u306ewell\u306b50\u03bcl\/well\u3067\u6dfb\u52a0\u3059\u308b\u3002\n<li>4\u03bcg\/ml\u306b\u5e0c\u91c8\u3057\u305fActinomycin D\u6eb6\u6db2\u309250\u03bcl\/well\u30674)\u306ewell\u306b\u6dfb\u52a0\u3059\u308b\u3002\n<li>37\u2103, CO<sub>2<\/sub> 5%\u306e\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30bf\u30fc\u306718\u6642\u9593\u57f9\u990a\u3059\u308b\u3002\n<\/ol>\n<p>(3)\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u67d3\u8272\u3000\uff08\u6bd2\u6027\u8a66\u9a13\uff08MTT\u6cd5\u7b49\uff09\u3067\u3082\u6e2c\u5b9a\u53ef\u80fd\uff09<\/p>\n<ol class=\"list_parentheses\">\n<li>\u57f9\u990a\u6db2\u3092\u9664\u304f\u3002\n<li>PBS(+)\u3092200\u03bcl\/well\u52a0\u3048\u308b\u3002\n<li>2\uff5e10\u5206\u653e\u7f6e\u3059\u308b\u3002\n<li>PBS(+)\u3092\u6368\u3066\u308b\u3002\n<li>10% \u4e2d\u6027\u7de9\u885d\u30db\u30eb\u30de\u30ea\u30f3\u6eb6\u6db2\u3092100\u03bcl\/well\u52a0\u3048\u3001\u5ba4\u6e29\u306730\u5206\u4ee5\u4e0a\u7f6e\u304f\u3002\uff08\u30db\u30eb\u30de\u30ea\u30f3\u6eb6\u6db2\u306f\u4e7e\u304d\u3084\u3059\u3044\u306e\u3067\u3001\u4e7e\u304b\u306a\u3044\u3088\u3046\u306b\u6ce8\u610f\u3059\u308b\u3002\uff09\n<li>\u30db\u30eb\u30de\u30ea\u30f3\u6eb6\u6db2\u3092\u9664\u304d\u3001PBS(+)\u3092100\u03bcl\/well\u52a0\u3048\u308b\u3002\n<li>PBS(+)\u3092\u6368\u3066\u308b\u3002\n<li>0.4% \u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u6eb6\u6db2\u309280\u03bcl\/well\u52a0\u3048\u3001\u5ba4\u6e29\u306715\uff5e30\u5206\u7f6e\u304f\u3002\n<li>\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u6eb6\u6db2\u3092\u53d6\u308a\u9664\u304d\u3001\u826f\u304f\u6c34\u6d17\u3059\u308b\u3002\n<li>\u4f0f\u305b\u3066\u4e7e\u304b\u3059\u3002\n<\/ol>\n<p>(4) \u6e2c\u5b9a<\/p>\n<ol class=\"list_parentheses\">\n<li>70\uff05\u30e1\u30bf\u30ce\u30fc\u30eb\u6eb6\u6db2\u3092200\u03bcl\/well\u52a0\u3048\u3001\u5ba4\u6e29\u30675\u5206\u7f6e\u304f\u3002\n<li>\u62bd\u51fa\u3055\u308c\u305f\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u304c\u5747\u4e00\u306b\u306a\u308b\u3088\u3046\u306b\u8efd\u304f\u63fa\u3059\u308b\u3002\n<li>595nm\uff08Single\uff09\u306e\u5438\u5149\u5ea6\u3092\u6e2c\u5b9a\u3002\n<\/ol>\n<p><strong>3.\u5224\u5b9a<\/strong><br \/>\nTNF\u03b1\u3092\u6dfb\u52a0\u3057\u3066\u3044\u306a\u3044\u30a6\u30a7\u30eb\u3092\u3001\u30b3\u30f3\u30c8\u30ed\u30fc\u30eb\u3068\u3057\u305f\u3002\u30b3\u30f3\u30c8\u30ed\u30fc\u30eb\u3092100\uff05\u3068\u3057\u305f\u3068\u304d\u306e\u3001TNF\u03b1\uff08\u307e\u305f\u306f\u691c\u4f53\uff09\u3092\u6dfb\u52a0\u3057\u305f\u30a6\u30a7\u30eb\u306e\u751f\u7d30\u80de\u6570\uff08\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u3067\u67d3\u8272\u3055\u308c\u305f\u7d30\u80de\uff09\u306e\u5272\u5408\uff08\uff05\uff09\u3092\u4e0b\u8a18\u30b0\u30e9\u30d5\u306b\u793a\u3057\u305f\u3002<\/p>\n<p>RCB1422\u3068RCB2619\u306f\u3001TNF\u03b1\u6dfb\u52a0\u3059\u308b\u3053\u3068\u306b\u3088\u308a\u3001\u6fc3\u5ea6\u4f9d\u5b58\u7684\u306b\u7d30\u80de\u304c\u6b7b\u6ec5\u3057\u305f\u3002<br \/>\nRCB1451\u306f\u3001100ng\/ml\u306eTNF\u03b1\u3092\u6dfb\u52a0\u3057\u3066\u3082\u3001\u7d30\u80de\u306f\u6b7b\u6ec5\u3057\u306a\u304b\u3063\u305f\u3002<\/p>\n<p><img decoding=\"async\" src=\"\/ja\/wp-content\/uploads\/images\/bioassay.png\"><\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>L929\u304cTNF\u03b1\u306b\u3088\u3063\u3066\u969c\u5bb3\u3092\u53d7\u3051\u308b\u6027\u8cea\u3092\u5229\u7528\u3057\u3066\u3001TNF\u03b1\u611f\u53d7\u6027\u8a66\u9a13\u3092\u884c\u3046\u65b9\u6cd5\u3067\u3059\u3002\u4fdd\u6709\u3057\u3066\u3044\u308b3\u682a\u306eL929\u7d30\u80de\uff08RCB1422\u3001RCB1451\u3001RCB2619\uff09\u3092\u4f7f\u7528\u3057\u3001\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u3092\u7528\u3044\u3066\u611f\u53d7\u6027\u306e\u9055\u3044\u3092\u6bd4\u8f03\u3059\u308b\u65b9\u6cd5\u3092\u3054\u7d39\u4ecb\u3057\u307e\u3059\u3002 *\u30d0\u30a4\u30aa\u30a2\u30c3\u30bb\u30a4\u306b\u3064\u3044\u3066 \u3053\u306e\u65b9\u6cd5\u3067\u65e2\u77e5\u306eTNF\u03b1\u3092\u7528\u3044\u3066\u691c\u91cf\u7dda\uff08\u30af\u30ea\u30b9\u30bf\u30eb\u30d0\u30a4\u30aa\u30ec\u30c3\u30c8\u3067\u67d3\u8272\u3055\u308c\u305f\u5272\u5408\uff09\u3092\u4f5c\u6210\u3057\u305f\u5f8c\u3001\u691c\u4f53\u306e\u5024\u3068\u691c\u91cf\u7dda\u3092\u6bd4\u3079\u308b\u3053\u3068 [&hellip;]<\/p>\n","protected":false},"author":8,"featured_media":0,"parent":1830,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_seopress_robots_primary_cat":"","_seopress_titles_title":"","_seopress_titles_desc":"","_seopress_robots_index":"","_mc_calendar":[],"footnotes":"","_wp_rev_ctl_limit":""},"class_list":["post-3399","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3399","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/comments?post=3399"}],"version-history":[{"count":9,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3399\/revisions"}],"predecessor-version":[{"id":9835,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3399\/revisions\/9835"}],"up":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/1830"}],"wp:attachment":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/media?parent=3399"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}