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字句 7 字句について (Lexical Convention) ECMAScript プログラムのソーステキストは最初に、トークン、行終端子、コメントあるいは空白のいずれかである入力要素のシーケンスに変換される。ソーステキストは、次の入力要素としてできるだけ長い文字シーケンスを繰り返しとって、左から右へと走査される。 字句文法には 2 つの目標記号がある。 InputElementDiv 記号は、除法演算子 (/) また除法代入演算子 (/=) が許される構文的文法コンテキストで使用される。 InputElementRegExp 記号は他の構文的文法コンテキストで使用される。 NOTE 構文的な文法内に、除算および RegularExpressionLiteral の両方が構文的文法によって許されるコンテキストが存在する; だが、そういう場合字句文法は InputElementDiv 目標記号を使用するので、そのコンテキスト中でスラッシュ開始は正規表現リテラル開始とは認められない。代替手段として、一つは括弧で正規表現リテラルを囲んでよい。 Syntax InputElementDiv WhiteSpace LineTerminator Comment Token DivPunctuator InputElementRegExp WhiteSpace LineTerminator Comment Token RegularExpressionLiteral 7.1 Unicode 制御文字 (Unicode Format-Control Characters) フォーマット制御のための (マークアップ言語のような) 上位手順がないので、 Unicode 形式制御文字 (例 LEFT-TO-RIGHT MARK や RIGHT-TO-LEFT MARK のような、 Unicode 文字データベース (Unicode Character Database) における "Cf" カテゴリの文字) という制御コードでテキストの範囲のフォーマットを制御する。編集を容易にしまた表示するため、ソーステキスト内でのこれらの許可は有益である。 フォーマット制御文字は ECMAScript プログラムのソーステキストのどの場所に出現してもよい。これらの文字は、字句文法を適用する前にソーステキストから取り除かれる。文字列と正規表現リテラルの処理前にこれらの文字が取り除かれるので、文字列、正規表現内に Unicode 制御文字を入れるには Unicode エスケープシーケンス (セクション 7.6) を使用しなければならない。 7.2 空白 (White Space) 空白文字は、ソーステキストの可読性を向上させ、そしてトークン (不可分の字句単位) を互いに分離させるために使用され、それ以上の意味はない。空白は 2 つのトークンの間、また文字列の中に出現できる(リテラル文字列値の一部を形成する意味のある文字と考えられる)。だが、トークンの中に出現することは出来ない。 次の文字が空白と考えられる Code Point ValueNameFormal Name \u0009Tab TAB \u000BVertical Tab VT \u000CForm Feed FF \u0020Space SP \u00A0No-break space NBSP Other category "Zs"その他の Unicode "space separator" USP Syntax WhiteSpace TAB VT FF SP NBSP USP 7.3 行終端子 (Line Terminators) 空白文字のように、行終端子はソーステキストの可読性を向上させ、そしてトークン (不可分の字句単位) を互いに分離させるために使用される。しかし、空白文字とは違い、行終端子は構文的文法の振る舞いに影響を与える。一般に、行終端子は 2 のトークンの間に出現する。だが構文的文法によって隠される場所がある。行終端子はトークンの中に出現することは出来ない。 not even a string. 行終端子は自動セミコロン挿入の処理にも影響を与える (セクション 7.9)。 空白は 2 つのトークンの間、また文字列の中に出現できる(リテラル文字列値の一部を形成する意味のある文字と考えられる)。だが、トークンの中に出現することは出来ない。 次の文字が行終端子と考えられる Code Point ValueNameFormal Name \u000ALine Feed LF \u000DCarriage Return CR \u2028Line separator LS \u2029Paragraph separator PS Syntax LineTerminator LF CR LS PS 7.4 コメント (Comments) Description コメントは、 1 行または複数行になりうる。複数行のコメントはネストできない。 1 行コメントは LineTerminator 以外の任意の文字を含むことが可能であること、またトークンは常に可能な長さである一般規則により、 1 行コメントは常に、マーカー // からその行の終わりまでの全ての文字で構成される。しかし、行末の LineTerminator は 1 行コメントの内容とはみなされない; それは字句文法によって別々に認識され、構文文法の入力要素のストリームの一部となる。この点がとても重要である。というのは、これは 1 行コメントの有無が自動セミコロン挿入 (セクション 7.9) に影響を与えないということを暗に意味するからである。 コメントは空白のように振る舞い、捨てられるが、 MultiLineComment が行終端子文字を含む場合、構文文法による解析を目的としてコメント全体が LineTerminator と考えられる、 Syntax Comment MultiLineComment SingleLineComment MultiLineComment /* MultiLineCommentCharsopt */ MultiLineCommentChars MultiLineNotAsteriskChar MultiLineCommentCharsopt PostAsteriskCommentCharsopt PostAsteriskCommentChars MultiLineNotForwardSlashOrAsteriskChar MultiLineCommentCharsopt PostAsteriskCommentCharsopt MultiLineNotAsteriskChar SourceCharacter but not asterisk * MultiLineNotForwardSlashOrAsteriskChar SourceCharacter but not forward-slash / or asterisk * SingleLineComment SingleLineCommentChars SingleLineCommentChar SingleLineCommentCharsopt SingleLineCommentChar SourceCharacter but not LineTerminator 7.5 トークン (Tokens) Syntax Token ReservedWord Identifier Punctuator NumericLiteral StringLiteral 7.5.1 予約語 (Reserved Words) Description 予約語を識別子として使うことはできない。 Syntax ReservedWord Keyword FutureReservedWord NullLiteral BooleanLiteral 7.5.2 キーワード (Keywords) 次のトークンは ECMAScript キーワードであり、 ECMAScript プログラム中で識別子に使うことはできない。 Syntax Keyword one of break case catch continue default delete do else finally for function if in instanceof new return switch this throw try typeof var void while with 7.5.3 将来の予約語 (Future Reserved Words) 次の語は提出された拡張の中でキーワードとして使用され、その拡張が将来採択される可能性を考え、予約される。 Syntax FutureReservedWord one of abstract boolean byte char class const debugger double enum export extends final float goto implements import int interface long native package private protected public short static super synchronized throws transient volatile 7.6 識別子 (Identifiers) Description 識別子は、若干の修正を加えた Unicode 標準 (バージョン 3.0 のセクション 5.16) で与えられる文法に従い解釈される。文法は Unicode 標準により指定される文字カテゴリの規範、及び参考情報の両方に基づいている。 Unicode 標準のバージョン 2.1 に指定されるカテゴリの文字は、全ての適合 ECMAScript 実装によってそれらのカテゴリ内として扱われなければならない; しかしながら、適合 ECMAScript 実装は、 Unicode の後のバージョンでのカテゴリ割り当てに基づく追加の合法的な識別子文字を許可してよい。 この標準は Unicode 標準に与えられる文法からある変更を指定する; ドル記号($) 及びアンダースコア (_) は識別子中のどこでも許される。ドル記号は機械的に生成されるコード中のみの使用を意図される。 Unicode エスケープシーケンスは識別子中でも許され、それは UnicodeEscapeSequence (セクション 7.8.4) の CV により算出されるように、識別子中のその場所に一文字を寄与する。 UnicodeEscapeSequence を使って識別子に不正な文字を設定することはできない。言い換えれば、\ UnicodeEscapeSequence シーケンスが UnicodeEscapeSequence の CV に置換されたら、結果は依然として有効な識別子で、正確に元の Identifier と文字の同じシーケンスなければならない。 Unicode 標準に従い規準的に等価である 2 つの識別子は、コードポイントの厳密に同じシーケンスによって表されない限りは等しくない(言い換えれば、適合 ECMAScript 実装は、識別子上でビット比較を行うことのみを要求される)。入ってくるソーステキストがコンパイラに行く前に正規化形式 C に変換されることを意図する。 Syntax Identifier ReservedWord でない IdentifierName IdentifierName IdentifierStart IdentifierName IdentifierPart IdentifierStart UnicodeLetter $ _ \ UnicodeEscapeSequence IdentifierPart IdentifierStart UnicodeCombiningMark UnicodeDigit UnicodeConnectorPunctuation \ UnicodeEscapeSequence UnicodeLetter Unicode カテゴリ "Uppercase letter (Lu)", "Lowercase letter (Ll)", "Titlecase letter (Lt)", "Modifier letter (Lm)", "Other letter (Lo)", "Letter number (Nl)" 内の任意の文字 UnicodeCombiningMark Unicode カテゴリ "Non-spacing mark (Mn)" または "Combining spacing mark (Mc)" 内の任意の文字 UnicodeDigit Unicode カテゴリ "Decimal number (Nd)" 内の任意の文字 UnicodeConnectorPunctuation Unicode カテゴリ "Connector punctuation (Pc)" 内の任意の文字 UnicodeEscapeSequence セクション 7.8.4 を参照。 HexDigit one of 0 1 2 3 4 5 6 7 8 9 a b c d e f A B C D E F 7.7 区切り子 (Punctuators) Syntax Punctuator one of { } ( ) [ ] . ; , = = == != === !== + - * % ++ -- | ^ ! ~ || ? = += -= *= %= = = = = |= ^= DivPunctuator one of / /= 7.8 リテラル (Literals) Syntax Literal NullLiteral BooleanLiteral NumericLiteral StringLiteral 7.8.1 Null リテラル (Null Literals) Syntax NullLiteral null Semantics null リテラルの値 null は、単独の Null 型の値で null と呼ばれる。 7.8.2 Boolean リテラル (Boolean Literals) Syntax BooleanLiteral true false Semantics Boolean リテラルの値 true は、 Boolean 型の値で true と呼ばれる。 Boolean リテラルの値 false は、Boolean 型の値で false と呼ばれる。 7.8.3 数値リテラル (Numeric Literals) Syntax NumericLiteral DecimalLiteral HexIntegerLiteral DecimalLiteral DecimalIntegerLiteral . DecimalDigitsopt ExponentPartopt . DecimalDigits ExponentPartopt DecimalIntegerLiteral ExponentPartopt DecimalIntegerLiteral 0 NonZeroDigit DecimalDigitsopt DecimalDigits DecimalDigit DecimalDigits DecimalDigit DecimalDigit one of 0 1 2 3 4 5 6 7 8 9 NonZeroDigit one of 1 2 3 4 5 6 7 8 9 ExponentPart ExponentIndicator SignedInteger ExponentIndicator one of e E SignedInteger DecimalDigits DecimalDigits DecimalDigits HexIntegerLiteral 0x HexDigit 0X HexDigit HexIntegerLiteral HexDigit NumericLiteral に直接続くソース文字は、けして IdentifierStart や DecimalDegit ではない。 NOTE 例えば 3in はエラーであり、 3 と in の 2 つの入力要素とはならない。 Semantics 数値リテラルは、 Number 型の値を表す。この値は 2 ステップで判断される まず、数学値 (MV) がリテラルから派生する; 次に、この数学値が、下に述べるように丸められる。 NumericLiteral DecimalLiteral の数学値は、 DecimalLiteral の数学値である。 NumericLiteral HexIntegerLiteral の数学値は、 HexIntegerLiteral の数学値である。 DecimalLiteral DecimalIntegerLiteral . の数学値は、 DecimalIntegerLiteral の数学値である。 DecimalLiteral DecimalIntegerLiteral . DecimalDigits の数学値は、 n を DecimalDigits の文字数として、 DecimalIntegerLiteral の数学値 + (DecimalDigits の数学値 × 10-n) である。 DecimalLiteral DecimalIntegerLiteral . ExponentPart の数学値は、 e を ExponentPart の数学値として DecimalIntegerLiteral の数学値 × 10e である。 DecimalLiteral DecimalIntegerLiteral . DecimalDigits ExponentPart の数学値は、 n を DecimalDigit の文字数、 e を ExponenetPart の数学値として、 (DecimalIntegerLiteral の数学値 + (DecimalDigits の数学値 × 10-n)) × 10e である。 DecimalLiteral . DecimalDigits の数学値は、 n を DecimalDigits の文字数として、 DecimalDigits の数学値 × 10-n である。 DecimalLiteral . DecimalDigits ExponentPart の数学値は、 n を DecimalDigits の文字数、 e を ExponenetPart の数学値として、 DecimalDigits の数学値の 10e-n である。 DecimalLiteral DecimalIntegerLiteral の数学値は、 DecimalIntegerLiteral の数学値である。 DecimalLiteral DecimalIntegerLiteral ExponentPart の数学値は、 e を ExponenetPart の数学値として、 DecimalIntegerLiteral の数学値 × 10e である。 DecimalIntegerLiteral 0 の数学値は、 0 である。 DecimalIntegerLiteral NonZeroDigit DecimalDigits の数学値は、 n を DecimalDigits の文字数として、 (NonZeroDigit の数学値の × 10n) + DecimalDigits の数学値である。 DecimalDigits DecimalDigit の数学値は、 DecimalDigit の数学値である。 DecimalDigits DecimalDigits DecimalDigit の数学値は、 (DecimalDigits の数学値 × 10) + DecimalDigit の数学値である。 ExponentPart ExponentIndicator SignedInteger の数学値は、 SignedInteger の数学値である。 SignedInteger DecimalDigits の数学値は、 DecimalDigits の数学値である。 SignedInteger + DecimalDigits の数学値は、 DecimalDigits の数学値である。 SignedInteger - DecimalDigits の数学値は、負の DecimalDigits の数学値である。 DecimalDigit 0 または HexDigit 0 の数学値は、 0 である。 DecimalDigit 1 または NonZeroDigit 1 または HexDigit 1 の数学値は、 1 である。 DecimalDigit 2 または NonZeroDigit 2 または HexDigit 2 の数学値は、 2 である。 DecimalDigit 3 または NonZeroDigit 3 または HexDigit 3 の数学値は、 3 である。 DecimalDigit 4 または NonZeroDigit 4 または HexDigit 4 の数学値は、 4 である。 DecimalDigit 5 または NonZeroDigit 5 または HexDigit 5 の数学値は、 5 である。 DecimalDigit 6 または NonZeroDigit 6 または HexDigit 6 の数学値は、 6 である。 DecimalDigit 7 または NonZeroDigit 7 または HexDigit 7 の数学値は、 7 である。 DecimalDigit 8 または NonZeroDigit 8 または HexDigit 8 の数学値は、 8 である。 DecimalDigit 9 または NonZeroDigit 9 または HexDigit 9 の数学値は、 9 である。 HexDigit a または HexDigit A の数学値は、 10 である。 HexDigit b または HexDigit B の数学値は、 11 である。 HexDigit c または HexDigit C の数学値は、 12 である。 HexDigit d または HexDigit D の数学値は、 13 である。 HexDigit e または HexDigit E の数学値は、 14 である。 HexDigit f または HexDigit F の数学値は、 15 である。 HexIntegerLiteral 0x HexDigit の数学値は、 HexDigit の数学値である。 HexIntegerLiteral 0X HexDigit の数学値は、 HexDigit の数学値である。 HexIntegerLiteral HexIntegerLiteral HexDigit の数学値は、 (HexIntegerLiteral の数学値 × 16) + HexDigit の数学値である。 一旦数値リテラルの厳密な数学値が決定されたら、 Number 型の値に丸められる。数学値が 0 ならば、丸められる値は +0 である; そうではなく、リテラルが DecimalLiteral でなくそのリテラルが 20 を超える有効数字数\でなければ、数値が 20 番目以降の各有効数字を数字 0 へ置換して生成されるリテラルの数学値の数値、または 20 番目以降の各有効数字を数字 0 に置換して生成されるリテラルの数学値の数値、そして 20 番目の有効数字の位置のリテラルの増加、のいずれかでありえる場合、丸められる値は (セクション 8.5 で定義される意味の) 数学値の数値でなければならない。数字はそれが ExponentPart の一部\でなく次の二点のどちらかであれば有効数字 (significant) である。 0 でない。 その左側が 0 以外の数字でその右側が 0 以外の数字で ExponentPart 内でない。 7.8.4 文字列リテラル (String Literals) 文字列リテラルは、単引用符または二重引用符で囲まれた 0 個以上の文字である。各文字はエスケープシーケンスによって表されてもよい。 Syntax StringLiteral " DoubleStringCharactersopt " SingleStringCharactersopt DoubleStringCharacters DoubleStringCharacter DoubleStringCharactersopt SingleStringCharacters SingleStringCharacter SingleStringCharactersopt DoubleStringCharacter SourceCharacter but not double-quote " or backslash \ or LineTerminator \ EscapeSequence SingleStringCharacter SourceCharacter but not single-quote or backslash \ or LineTerminator \ EscapeSequence EscapeSequence CharacterEscapeSequence 0 [lookahead ∉ DecimalDigit] HexEscapeSequence UnicodeEscapeSequence CharacterEscapeSequence SingleEscapeCharacter NonEscapeCharacter SingleEscapeCharacter one of " \ b f n r t v NonEscapeCharacter SourceCharacter but not EscapeCharacter or LineTerminator EscapeCharacter SingleEscapeCharacter DecimalDigit x u HexEscapeSequence x HexDigit HexDigit UnicodeEscapeSequence u HexDigit HexDigit HexDigit HexDigit 非終端記号 HexDigit の定義は、セクション 7.8.3 に与える。 SourceCharacter はセクション 2 及び 6 で述べる。 文字列リテラルは String 型の値を表す。リテラルの文字列値 (SV) は、文字列値の様々な部分に寄与する文字値 (CV) に関して記述される。この処理の一部として、文字列リテラル内部のいくつかの文字は、セクション 7.8.3 に述べる数学値 (MV) を持つものとして解釈される。 StringLiteral "" の文字列値は、空の文字シーケンスである。 StringLiteral の文字列値は、空の文字シーケンスである。 StringLiteral " DoubleStringCharacters " の文字列値は、DoubleStringCharacters の文字列値である。 StringLiteral SingleStringCharacters の文字列値は、SingleStringCharacters の文字列値である。 DoubleStringCharacters DoubleStringCharacter の文字列値は、1 文字のシーケンスで、 DoubleStringCharacter の文字値である。 DoubleStringCharacters DoubleStringCharacter DoubleStringCharacters の文字列値は、順に DoubleStringCharacters の文字列値内の全ての文字の続く DoubleStringCharacter の文字値のシーケンスである。 SingleStringCharacters SingleStringCharacter の文字列値は、1 文字のシーケンスで、 SingleStringCharacter の文字値である。 SingleStringCharacters SingleStringCharacter SingleStringCharacters の文字列値は、順に SingleStringCharacters の文字列値内の全ての文字の続く SingleStringCharacter の文字値のシーケンスである。 DoubleStringCharacter SourceCharacter but not 二重引用符 " or バックスラッシュ \ or LineTerminator の文字値は、 SourceCharacter 文字それ自身である。 DoubleStringCharacter \ EscapeSequence の文字値は、 EscapeSequence の文字値である。 SingleStringCharacter SourceCharacter but not 単引用符 or バッククラッシュ \ or LineTerminator の文字値は、 SourceCharacter 文字それ自身である。 SingleStringCharacter \ EscapeSequence の文字値は、 EscapeSequence の文字値である。 EscapeSequence CharacterEscapeSequence の文字値は、 CharacterEscapeSequence の文字値である。 EscapeSequence 0 [lookahead ∉ DecimalDigit] の文字値は、 NUL 文字 (Unicode 値 0000) である。 EscapeSequence HexEscapeSequence の文字値は、 HexEscapeSequence の文字値である。 EscapeSequence UnicodeEscapeSequence の文字値は、 UnicodeEscapeSequence の文字値である。 CharacterEscapeSequence SingleEscapeCharacter の文字値は、次の表に従い SingleEscapeCharacter に決定されるコードポイント値の文字である Escape SequenceCode Point ValueNameSymbol \b\u0008backspace BS \t\u0009horizontal tab HT \n\u000Aline feed (new line) LF \v\u000Bvertical tab VT \f\u000Cform feed FF \r\u000Dcarriage return CR \"\u0022double quote" \ \u0027single quote \\\u005Cbackslash\ CharacterEscapeSequence NonEscapeCharacter の文字値は、 NonEscapeCharacter の文字値である。 NonEscapeCharacter SourceCharacter but not EscapeCharacter or LineTerminator の文字値は、 SourceCharacter 文字それ自身である。 HexEscapeSequence x HexDigit HexDigit の文字値は、コードポイント値が (16 × 最初の HexDigit の数学値) + 2番目の HexDigit の数学値 である文字である。 UnicodeEscapeSequence u HexDigit HexDigit HexDigit HexDigit の文字値は、コードポイント値が (4096 (すなわち 163) × 最初の HexDigit の数学値) + (256 (すなわち 162) × 2 番目の HexDigit の数学値) + (16 × 3 番目の HexDigit の数学値) + 4 番目の HexDigit の数学値 である文字である。 NOTE 文字 LineTerminator は、それにバックスラッシュ \ を先行させても、文字列リテラル内には出現できない。 行終端子文字を文字列リテラルの文字列地の一部に置く妥当な方法は、 \n や \u000A のようなエスケープシーケンスを用いることである。 7.8.5 正規表現リテラル (Regular Expression Literals) 正規表現リテラルは走査時に RegExp オブジェクト (セクション 15.10) に変換される入力要素である。オブジェクトはそれを含むプログラムまた関数の評価の開始前に生成される。リテラル評価はそのオブジェクトへの参照を生成する; それは新規オブジェクト生成はしない。プログラム内の 2 つの正規表現リテラルは、2 つのリテラルの内容がまったく同じであっても、互いに === として比較しない正規表現オブジェクトに評価する。 RegExp オブジェクトは、 new RegExp (セクション 15.10.4) や関数としての RegExp コンストラクタ呼出し (セクション 15.10.3) によって実行時に生成されてもよい。 下の生成規則は正規表現リテラルの構文を記述し、正規表現リテラルの終了の検出に入力要素走査から使用される。 RegularExpressionBody 及び RegularExpressionFlags を構成する文字の文字列は、それ自身がより厳重な文法でそれらを解釈する正規表現コンストラクタに、未解釈で渡される。実装は正規表現コンストラクタの文法を拡張してよいが、 RegularExpressionBody 及び RegularExpressionFlags 生成規則、またはこれらの生成規則によって使用される生成規則を拡張すべきではない。 Syntax RegularExpressionLiteral / RegularExpressionBody / RegularExpressionFlags RegularExpressionBody RegularExpressionFirstChar RegularExpressionChars RegularExpressionChars [empty] RegularExpressionChars RegularExpressionChar RegularExpressionFirstChar NonTerminator but not * or \ or / BackslashSequence RegularExpressionChar NonTerminator but not \ or / BackslashSequence BackslashSequence \ NonTerminator NonTerminator SourceCharacter but not LineTerminator RegularExpressionFlags [empty] RegularExpressionFlags IdentifierPart NOTE 正規表現リテラルは空にはならない; 空の正規表現リテラルをあらわす代わりに、文字 // は 1 行コメントを開始する。空の正規表現を指定するには、 /(? )/ を用いる。 Semantics 正規表現リテラルは Object 型の値を表す。この値は 2 ステップで決定される まず、正規表現の RegularExpressionBody 及び RegularExpressionFlags 生成規則拡張を構成する文字が、それぞれ 2 つの文字列の Pattern と Flags に解析されずに集められる。そして new RegExp コンストラクタが 2 つの引数 Pattern と Flags で呼出され、結果は RegularExpressionLiteral の値となる。 new RegExp 呼出しがエラーを生成するならば、実装は、自由裁量で、プログラム走査中に直ちにエラー報告してもよいし、プログラム実行中に正規表現リテラルが評価されるまでエラーを延期してもよい。 7.9 自動セミコロン挿入 (Automatic Semicolon Insertion) ある種の ECMAScript 文 (空文 (empty statement), 変数文 (variable statement), 式文 (expression statement), do-while 文 (do-while statement), continue 文 (continue statement), break 文 (break statement), return 文 (return statement), turhow 文 (throw statement)) はセミコロンで終了しなければならない。そのようなセミコロンは、ソーステキスト内に常に明示的に出現してよい。しかしながら、簡単にするために、ある位置ではソーステキストからセミコロンを省略してよい。この位置は、それらの位置のソースコードトークンのストリームにセミコロンが自動的に挿入されるという言葉で説明される。 7.9.1 自動セミコロン挿入規則 (Rules of Automatic Semicolon Insertion) プログラムが左から右へと解析され、文法の任意の生成規則によって許可されないトークン (違反トークン (offending token) と呼ばれる) に遭遇するとき、次の条件に 1 つ以上当てはまる場合、違反トークンの前にセミコロンが自動的に挿入される。 1. 違反トークンが、少なくとも 1 つの LineTerminator で、前のトークンから分離されている。 2. 違反トークンが、 } である。 プログラムが左から右へと解析され、トークンの入力ストリームが終了し、解析器が入力トークンストリームを一つの完全な ECMAScript Program として解析できないとき、入力ストリームの末尾にセミコロンが自動的に挿入される。 プログラムが左から右へと解析され、文法の任意の生成規則によって許可されないトークンに遭遇するが、その生成規則が限定生成規則 (resutricted production) であり、限定生成規則内部\で注記 "[no LineTerminator here]" にすぐ続く終端記号または非終端記号の最初のトークン (そのためこのようなトークンは限定トークン (restricted token) と呼ばれる) であり、そして限定トークンが少なくとも 1 つの LineTerminator で前のトークンから分離されているとき、限定トークンの前にセミコロンが自動的に挿入される。 とはいえ、以上の規則を上書きする条件を追加する セミコロンが結果として空文として解析される場合、またセミコロンが for 文 (セクション 12.6.3) のヘッダ内の 2 つのセミコロンの一つになる場合は、セミコロンは自動的に挿入されない。 NOTE 文法における限定生成規則は下記のみである PostfixExpression LeftHandSideExpression [LineTerminator 無し] ++ LeftHandSideExpression [LineTerminator 無し] -- ContinueStatement continue [LineTerminator 無し] Identifieropt ; BreakStatement break [LineTerminator 無し] Identifieropt ; ReturnStatement return [LineTerminator 無し] Expressionopt ; ThrowStatement throw [LineTerminator 無し] Expression ; これらの限定生成規則の実際の効果は、次のとおりである 解析器がそれを後置演算子として扱う場所に ++, -- トークンが出現し、少なくとも一つ以上の LineTerminator が前のトークンと ++, -- トークンの間に出現するとき, セミコロンが ++, -- トークンの前に自動的に挿入される。 continue, break, return, throw トークンが出現し、次のトークンの前に LineTerminator が出現するとき、セミコロンは continue, break, return, throw トークンの後に自動的に挿入される。 ECMAScript プログラマへの結果的な実用的助言は 後置 ++ 演算子、また後置 -- 演算子は、項と同じ行に出現するべきである。 return 文、また throw 文中の Expression は、return トークンまた throw トークンと同じ行で開始するべきである。 break 文、また continue 文のラベルは、 break トークンまた continue トークンと同じ行にあるべきである。 7.9.2 自動セミコロン挿入例 (Examples of Automatic Semicolon Insertion) 次のソース { 1 2 } 3 は、ECMAScript 文法において、自動セミコロン挿入規則を以ってしても有効な文ではない。 対照的に、このソース { 1 2 } 3 もまた有効な ECMAScript 文ではないが、自動セミコロン挿入によって次のように変換される { 1 ;2 ;} 3; これは有効な ECMAScript 文である。 ソース for (a; b ) は有効な ECMAScript 文ではなく、自動セミコロン挿入によって変更されない。セミコロンが for 文のヘッダに要求されるからである。自動セミコロン挿入が for 文のヘッダ内の 2 つのセミコロンの 1 つを挿入することはない。 ソース return a + b は自動セミコロン挿入によって次のように変換される return; a + b; NOTE 式 a + b は、 return 文によって返される値として扱われない。return トークンから LineTerminator で分離されているからである。 ソース a = b c は自動セミコロン挿入によって次のように変換される a = b; c; NOTE トークン ++ は、変数 b に適用される後置演算子として扱われない。 b と ++ の間に LineTerminator があるからである。 ソース if (a b) else c = d は有効な ECMAScript 文ではなく、その位置に適用する文法の生成規則がないとしても、 else トークンの前への自動セミコロン挿入によって変更されない。自動セミコロン挿入が空文として解析されるからである。 ソース a = b + c (d + e).print() は自動セミコロン挿入によって変換されない。 2 行目の括弧式 () は関数呼び出しの引数リストとして解釈できるからである。 a = b + c(d + e).print() 代入文が左括弧で開始しなければならない状況では、自動セミコロン挿入を信頼するより、先行する文の末尾に明示的なセミコロンを提供する方が、プログラマにとってよい発想である。
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Release Note Release Date 5 Aug, 2009 Notes Cadencii requires ".NET Framework Runtime(version 2.0 or later)". Installer of this rumtime is available from the link below. .NET Framework Runtime Download .NET Framework 3.5 SP1 Cadencii can be launched with the latest version of mono. This enable you to use Cadencii with many platforms supported by mono. (Note Several functions using VOCALOID2 VSTi are not available in this case.) Mono is available from the link mono download Download Cadencii version 2.3.0 (634KB) How to get source codes Source code is available on SourceForege.JP. Please follow the instruction below for checking out the SourceForge.JP s SVN repository. svn checkout -r 401 http //svn.sourceforge.jp/svnroot/cadencii/branches/2.3 ./ These svn command is for checiking out "THIS" version of Cadencii. In order to get the latest source codes, please remove "-r" option.
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Ascent鯖 鯖プログラム開発元 http //www.ascentemu.com/ svn svn //svn.ascentemu.com/svn/ascent/ svn http //projects.emupedia.com/svn/ascent/ スクリプト GameMonkey svn http //projects.emupedia.com/svn/gmscripts/ DB開発元 Next-Chapter Project http //ncdb.info/ svn http //projects.emupedia.com/svn/ncdb/ 略称:NCDB GamingEVOLVED http //gamingevolved.org/ ショッピングモールが充実しています TerraNet http //wow.terra.net.ba/ Project Silvermoon http //projectsilvermoon.net/ 略称:TNPS License CCPL Operating System 32-bit MS Windows, Linux, MacOSX-PPC 開発言語:C++ ソースコード開示:○ 開発元からのWin32 Binaries配布:× DB:MySQL v5 Win32 Binaries 配布中 http //filebeam.com/folder/2633/ Ascent鯖を建てよう 最新の安定版2.0-STABLEで鯖を建ててみましょう ダウンロード 2_0_stable r1993.rar DBC_2_2_3.rar Maps222.rar Milestone 0.2.1.rar 解凍 2_0_stable r1993.rarをC \に解凍します DBC_2_2_3.rarをC \2_0_stable r1993に解凍します Maps222.rarをC \2_0_stable r1993\Mapsに解凍します Milestone 0.2.1.rar内の Milestone 0.2.1\Database\world.sqlをC \2_0_stable r1993に解凍します Ascent/Ascent@MySQLの作成 GRANT ALL PRIVILEGES ON *.* TO ascent@localhost IDENTIFIED BY ascent WITH GRANT OPTION; set password for ascent @ localhost = OLD_PASSWORD( ascent ); 以上の3行をmysql.exe等で入力します 各sqlファイルのインポート C \2_0_stable r1993\ImportDB.batを実行します test/test@ログインアカウントの作成 use ncdb_character; INSERT INTO `accounts` VALUES (1, test , test , az ,0, , , ,8); 以上の3行をmysql.exe等で入力します ascent.confの編集 同梱済 logonserver.confの編集 同梱済 エミュ鯖の起動 logonserver.exe ascent.exe の順に起動します realmlist.wtfの修正 C \Program Files\World of Warcraft\realmlist.wtfの修正 set realmlist localhost クライアントの起動 C \Program Files\World of Warcraft\WoW.exe を直接実行して起動します Account Name test Account Password test でログインします 以下おまけ スタックしてしまった時バインドポイントに移動させるSQL文 update characters SET positionX = bindpositionX , positionY = bindpositionY, positionZ = bindpositionZ , mapId = bindmapId, zoneId = bindzoneId, deathstate = 0 WHERE name = 救いたいキャラ名 ; export_accounts.bat export_accounts.bat import_accounts.bat import_accounts.bat ひとりでRaidZoneに入る方法 DBによっては縛りがデフォルトのままでひとりでは入れません DBを修正してしまいましょう update worldmap_info set type = 2 where type = 1 ; これで入れるようになります( ^ω^) GM AddOns AntrixTeleport http //skullsofsteel.de/downloads/addons/AntrixTeleport.rar /ATPで窓が開きます Antrix GM Addon LINKの最上段 svn //bitmonkey.homelinux.com/antrixgmaddon/tags/ svn //bitmonkey.homelinux.com/antrixgmaddon/trunk/ minimapにボタンが付きます Full GM Command List Banstick Commands [b] banchar Bans character x with or without reason unbanchar Unbans character x kick Kicks player from server paralyze Roots/Paralyzes the target. unparalyze Unroots/Unparalyzes the target. Chat Control [c] allowwhispers Allows whispers from player s while in gmon mode. blockwhispers Blocks whispers from player s while in gmon mode. Debug Commands [d] debug Main ‘debug’ command table. Usage .debug subcommand args infront No description entered. showreact No description entered. aimove No description entered. dist No description entered. face No description entered. moveinfo No description entered. setbytes No description entered. getbytes No description entered. unroot No description entered. root No description entered. landwalk No description entered. waterwalk No description entered. castspell .castspell spellid - Casts spell on target. castspellne .castspellne spellid - Casts spell on target (only plays animations, doesnt handle effects or range/facing/etc. celldelete USE WITH CAUTION! .celldelete YES - Removes everything in current cell from game and database. .celldelete YES YES removes everything in a range of 1cell. addrestxp .addrestxp - Adds x rest XP to player. generatename .generatename - Generates name for pet, etc. attackerinfo .attackerinfo - Shows selected mob/player s attacker s infomation. showattackers .showattackers - Shows selected mob/player s attacker on the minimap. aggrorange .aggrorange - Shows aggro Range of the selected Creature. knockback .knockback value - Knocks you back. fade .fade value - calls ModThreatModifyer(). threatMod .threatMod value - calls ModGeneratedThreatModifyer(). calcThreat .calcThreat dmg spellId - calculates threat. threatList .threatList - returns all AI_Targets of the selected Creature. gettptime grabs transporter travel time itempushresult sends item push result weather No description entered. setbit No description entered. setvalue No description entered. getpos No description entered. Battlegrounds Commands [e] battleground Main BG command table. Usage .battleground subcommand args setbgscore Teamid Score - Sets battleground score. 2 Arguments. startbg Starts current battleground match. pausebg Pauses current battleground match. bginfo Displays information about current battleground. battleground Shows BG Menu setworldstate var val - Var can be in hex. WS Value. playsound val . Val can be in hex. setbfstatus .setbfstatus – Not Yet Implemented. leave Leaves the current battleground. Extended Debug Commands? [f] killbyplayer Disconnects the player with name s . killbyaccount Disconnects the session with account name s . getrate Gets rate x . setrate Sets rate x . GM Ticket Commands [g] gmTicket Main GM ticket system command table. Usage .gmTicket subcommand args get Gets GM Ticket getId Gets GM Ticket by ID delId Deletes GM Ticket by ID Invincibility Command [j] invincible .invincible - Toggles INVINCIBILITY (mobs won t attack you) Invisibility Command [i] invisible .invisible - Toggles INVINCIBILITY and INVISIBILITY (mobs won t attack you and nobody can see you, but they can see your chat messages) Guild Commands [l] createguild No description entered. Modify commands [m] modify Main stat-modification command table. Usage .modify subcommand args hp Health Points/HP mana Mana Points/MP rage Rage Points energy Energy Points armor Armor holy Holy Resistance fire Fire Resistance nature Nature Resistance frost Frost Resistance shadow Shadow Resistance arcane Arcane Resistance damage Unit Damage Min/Max scale Size/Scale gold Gold/Money/Copper speed Movement Speed nativedisplayid Native Display ID displayid Display ID flags Unit Flags faction Faction Template dynamicflags Dynamic Flags talentpoints Talent Points loyalty Loyalty spirit Spirit boundingraidus Bounding Radius combatreach Combat Reach emotestate NPC Emote State More Modify Commands [m] createaccount .createaccount - Creates account. Format should be .createaccount username password email playerinfo .playerinfo - Displays informations about the selected character (account...) mount Mounts into modelid x. dismount Dismounts. start Teleport s you to a starting location levelup No description entered. additem No description entered. removeitem Removes item %u count %u. learn Learns spell unlearn Unlearns spell learnskill .learnskill skillid (optional) value maxvalue - Learns skill id skillid. advanceskill advanceskill skillid amount, optional, default = 1 - Advances skill line x times.. removeskill .removeskill skillid - Removes skill increaseweaponskill .increaseweaponskill count - Increase eqipped weapon skill x times (defaults to 1). removeauras Removes all auras from target setmotd Sets MOTD additemset Adds item set to inv. gotrig Warps to areatrigger id createinstance Creates instance on map map goinstance Joins instance instance x y z optional mapid exitinstance Exits current instance, return to entry point. dbreload Reloads some of the database tables spawnspiritguide Spawns a spirit guide (params 1 = horde, 0 = alliance) advanceallskills Advances all skills x points. unlockmovement Unlocks movement for player. modperiod Changes period of current transporter. npcfollow Sets npc to follow you follow Sets npc to not follow anything formationlink1 Sets formation master. formationlink2 Sets formation slave with distance and angle formationclear Removes formation from creature playall Plays a sound to the entire server. addipban Bans an ip address address/mask duration, 0=perm banaccounts Bans accounts name duration, 0=perm renamechar Renames character x to y. forcerenamechar Forces character x to rename his char next login getstanding Gets standing of faction %u. setstanding Sets standing of faction %u. Cheat Commands [m] cheat Main cheat command table. Usage .cheat subcommand args status Shows active cheats. taxi Enables all taxi nodes. cooldown Enables no cooldown cheat. casttime Enables no cast time cheat. power Disables mana consumption etc. god Sets god mode, prevents you from taking damage. fly Sets fly mode land Unsets fly mode explore Reveals the unexplored parts of the map. flyspeed Modifies fly speed. stack Enables aura stacking cheat. morph Morphs into model id x. demorph Demorphs from morphed model. Honor Modification Commands [m] honor Main honor system command table. Usage .honor subcommand args getpvprank Gets PVP Rank setpvprank Sets PVP Rank addpoints Adds x amount of honor points/currency addkills Adds x amount of honor kills globaldailyupdate Daily honor field moves singledailyupdate Daily honor field moves for selected player only pvpcredit Sends PVP credit packet, with specified rank and points Pet Commands [m] pet Main pet command table. Usage .pet subcommand args createpet Creates a pet with entry . renamepet Renames a pet to name . enablerename Enables pet rename. addspell Teaches pet spell . removespell Removes pet spell spell . NPC Commands [n] npc Main NPC command table. Usage .npc subcommand args vendoradditem Adds to vendor vendorremoveitem Removes from vendor. flags Changes NPC flags emote emote - Sets emote state run No description entered. addweapon No description entered. allowmove No description entered. addgrave No description entered. addspirit No description entered. faction No description entered. delete Deletes mob from db and world. info Displays NPC information guid Shows selected object guid addAgent .npc addAgent agent procEvent procChance procCount spellId spellType spelltargetType spellCooldown floatMisc1 Misc2 delAgent .npc delAgent procEvent spellId listAgent .npc listAgent reset resets npc health/dmg from temp table. export Exports the npc to a sql file say .npc say text - Makes selected mob say text text . yell .npc yell Text - Makes selected mob yell text text . come .npc come - Makes npc move to your position return .npc return - Returns ncp to spawnpoint. spawn .npc spawn - Spawns npc of entry id spawnlink .spawnlink sqlentry resetreputation .resetreputation - Resets reputation to start levels. (use on characters that were made before reputation fixes.) resetlevel .resetlevel - Resets all stats to level 1 of targeted player. DANGEROUS. resetspells .resetspells - Resets all spells to starting spells of targeted player. DANGEROUS. resettalents .resettalents - Resets all talents of targeted player to that of their current level. DANGEROUS. resetskills .resetskills - Resets all skills. GameObject Commands [o] gobject Main gameobject command table. Usage .gobject subcommand args select Selects the nearest GameObject to you delete Deletes selected GameObject spawn Spawns a GameObject by ID info Gives you informations about selected GO activate Activates/Opens the selected GO. enable Enables the selected GO for use. scale Sets scale of selected GO animprogress Sets anim progress export Exports the current GO selected statelink .statelink sqlentry, Links a GO state to a Npc Information Commands [p] gm Shows active GM s gps Shows Position info Server info uptime Shows server uptime Teleport (Recall)Commands [q] recall Main teleportation command table. Usage .recall subcommand args list List recall locations port Port to recalled location add Add recall location del Remove a recall location portplayer recall ports player Life and Death Commands [r] kill .kill - Kills selected unit. revive Revives you. 0, 0, 0}, reviveplr Revives player specified. Save Commands [s] save Save s your character saveall Save s all playing characters GM Tag Commands [t] fmoff Sets GM tag off gmon Sets GM tag on Universal Commands [u] Commands Shows Commands help Shows help for command announce Sends Msg To All wannounce Sends Widescreen Msg To All Extended Teleport Commands [v] appear Teleports to x s position. summon Summons x to your position worldport No description entered. Waypoint Commands [w] waypoint waypointCommandTable add Add wp at current pos show Show wp s for creature hide Hide wp s for creature delete Delete selected wp movehere Move to this wp flags Wp flags waittime Wait time at this wp emote Emote at this wp skin Skin at this wp change Change at this wp info Show info for wp movetype Movement type at wp generateNo description entered.Randomly generate wps save Save all waypoints deleteall Delete all waypoints Admin Commands [z] security No description entered. servershutdown Initiates server shutdown in x seconds. serverrestart Initiates server restart in x seconds. castall Makes all players online cast spell x .
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memo source 1 /* Licensed to the Apache Software Foundation (ASF) under one or more 2 * contributor license agreements. See the NOTICE file distributed with 3 * this work for additional information regarding copyright ownership. 4 * The ASF licenses this file to You under the Apache License, Version 2.0 5 * (the "License"); you may not use this file except in compliance with 6 * the License. You may obtain a copy of the License at 7 * 8 * http //www.apache.org/licenses/LICENSE-2.0 9 * 10 * Unless required by applicable law or agreed to in writing, software 11 * distributed under the License is distributed on an "AS IS" BASIS, 12 * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. 13 * See the License for the specific language governing permissions and 14 * limitations under the License. 15 */ 16 17 #ifndef APACHE_HTTP_MAIN_H 18 #define APACHE_HTTP_MAIN_H 19 20 #include "apr_optional.h" 21 22 /* AP_SERVER_BASEARGS is the command argument list parsed by http_main.c 23 * in apr_getopt() format. Use this for default ing args that the MPM 24 * can safely ignore and pass on from its rewrite_args() handler. 25 */ 26 #define AP_SERVER_BASEARGS "C c D d E e f vVlLtSh?X" 27 28 #ifdef __cplusplus 29 extern "C" { 30 #endif 31 32 /** 33 * @package Command line options 34 */ 35 36 /** The name of the Apache executable */ 37 AP_DECLARE_DATA extern const char *ap_server_argv0; 38 /** The global server s ServerRoot */ 39 AP_DECLARE_DATA extern const char *ap_server_root; 40 41 /* for -C, -c and -D switches */ 42 /** An array of all -C directives. These are processed before the server s 43 * config file */ 44 AP_DECLARE_DATA extern apr_array_header_t *ap_server_pre_read_config; 45 /** An array of all -c directives. These are processed after the server s 46 * config file */ 47 AP_DECLARE_DATA extern apr_array_header_t *ap_server_post_read_config; 48 /** An array of all -D defines on the command line. This allows people to 49 * effect the server based on command line options */ 50 AP_DECLARE_DATA extern apr_array_header_t *ap_server_config_defines; 51 52 APR_DECLARE_OPTIONAL_FN(int, ap_signal_server, (int *, apr_pool_t *)); 53 54 #ifdef __cplusplus 55 } 56 #endif 57 58 #endif /* !APACHE_HTTP_MAIN_H */
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Infantry Captain Contents 1 Tactics 2 History 3 Built From 3.1 Field Support Truck 4 Support Abilities 4.1 Forward Observation Officer Barrage 5 Squad Abilities 5.1 Victor Target 6 Squad Weapons 6.1 Webley Revolver Captain Squad Size 1 Capture Rate 1 Sup Threshold 0.35 Health 60 Sight 35 Pin Threshold 0.8 Cost 300 Detection 10/0 Recovery Rate 0.012 Hotkey C Population 1 Time 60 Retreat Modifier 0.5 Target Type infantry_heroic Upkeep 1.5024 Reinforce Cost 0.5 Critical Type infantry_heroic Squad Slots 2 Reinforce Time 2 Tactics The captain is an officer much like the Lieutenant. Unlike the Lieutenant, Captains do NOT give speed bonuses. Captains solely give defensive bonuses and quite good at that. He will give bonuses to an entire sector and receive veterancy from all units within. It is a good idea for Captains to pick up fallen enemy weapons, as their standard weapon is generally ineffective. History Captain is a rank in the British armed forces that is used in the Army, Royal Navy, and the Royal Marines. The title is used by two separate rank grades however. Royal Navy - the rank of Captain in the Royal Navy is at the NATO OF-5 grade, equivalent to a full Colonel in the Army and Royal Marines or a Group Captain in the RAF; and Army/Royal Marines in the Army and Royal Marines, Captain is the OF-2 grade and is parallel to a Lieutenant in the Royal Navy or a Flight Lieutenant in the RAF. Built From Field Support Truck [Expand][Hide] Health 750 Population 0 Cost 18530 Max Speed 3 Time 70 Target Type hqs_mobile Hotkey F Effects The Field Support Truck coordinates and constructs Stuart Light Tanks, Sappers, and Captains. ESee Vehicle Field Support Truck for details. Support Abilities Forward Observation Officer Barrage [Expand][Hide] Cost 150 Activation targeted Duration _ Target tp_position Recharge 30 Hotkey B Effects Lieutenants and Captains can call in an artillery barrage. ESee Ability Forward Observation Officer Barrage for details. Squad Abilities Victor Target [Expand][Hide] Cost Activation targeted Duration _ Target tp_any Recharge 180 Hotkey V Effects The Victor Target will order all heavy artillery pieces (25 Pounders and Priests) to fire simultaneously at the same point. Artillery rounds fired to bombard the Victor Target will ignore their usual range constraints. Artillery pieces ordered to Counterbattery or Overwatch an area will not join in the barrage. ESee Ability Victor Target for details. Squad Weapons Webley Revolver [Expand][Hide] Weapon Webley Revolver See Weapon Webley Revolver for details. Retrieved from http //coh-stats.com/Infantry Captain
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ActiveRecordとは ActiveRecordメソッド ActiveRecordメソッドを使用することで、様々な条件に応じてデータを取得できる。 (参考元) https //qiita.com/Hal_mai/items/babb19560ace072c99f5 (参考元) https //qiita.com/ryokky59/items/a1d0b4e86bacbd7ef6e8 new save create link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。 link_anchor plugin error 画像もしくは文字列を必ずどちらかを入力してください。{) new 指定したモデルと結びつく、テーブルのカラム名がキーとなったモデルクラス(ハッシュのようなもの)のインスタンスを生成する。 save newで生成したインスタンスを保存し、DBに反映させる。 create
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Vehicle Schwimmwagen Type 128 Contents 1 Schwimmwagen Type 128 Veterancy 2Info 3Tactics 4Built From 4.1 Panzer Elite Headquarters 5Doctrinal Abilities 6Vehicle Abilities 6.1 Vehicle Cover Schwimmwagen Type 128 Health 90 Max Speed 7.5 Sight 55 Cost 165 Acceleration 14 Detection 30/10 Time 25 Deceleration 9 Hotkey W Population 1 Rotation 120 Target Type sdkfz_22x Upkeep 2.688 Crush Human False Critical Type supply_truck Crush Mode None Rear Damage Enabled true Schwimmwagen Type 128 Veterancy [Expand][Hide] Received Damage Received Accuracy Maximum Speed Maximum Health 0.9 0.9 1.15 1.15 8 Vet-Exp Received Damage Received Accuracy Maximum Speed Maximum Health 0.95 0.95 1.05 1.15 22 Vet-Exp Received Damage Received Accuracy Maximum Speed Maximum Health 0.95 0.95 1.05 1.15 38 Vet-Exp Info The Panzer Elite Schwimmwagen Type 128 (Not to be confused with the Type 166 for Wehrmacht) is an alternative unit introduced in Tales of Valor. It can be chosen to swap in place with the Kettenkrad. The Schwimmwagen Type 128 is identical to the Kettenkrad, with the exception of having different abilities and being able to travel through water terrain faster. Like the Kettenkrad, it can gain one of three abilities, depending on the doctrine chosen. Scorched Earth grants the Incendiary Trap ability, Luftwaffe grants the Mark Target ability, and Tank Destroyer grants the Mine Drop ability. Incendiary Traps are incendiary mines that can be laid quickly by the Schwimmwagen. It costs 15 munitions and is only triggered by enemy infantry. When it explodes, it creates a large area of flames that deal damage over time. The damage is statistically identical to a single Incendiary Mortar round. Mark Target reveals the target, even through the fog of war, and increases its received accuracy by x1.2, for a duration of 20 seconds. Mine Drop drops a normal mine, and it costs more munitions than normal (35 vs 25). Schwimmwagens of both varieties suffer less movement speed penalties when travelling through water terrain, as they have a 0.8 (80%) modifier compared to 0.3 (30%) for most vehicles. Tactics The Schwimmwagen Type 128, like the Kettenkrad, has no weapons and is used mainly for capturing points and reconnaissance. Incendiary Trap can be used as anti-infantry mines. Since they are incendiary-based, they should be placed in spots where enemy infantry is likely to stand around in. For example, near resource points, or the entrances of buildings, where the flames can spread out toward the garrison and still damage them. Mark Target has a very short 10 second cooldown time and should be used as much as possible if the Schwimmwagen is on reconnaissance duties. This ability is handy for tracking or killing units, including camouflaged ones like Snipers, as it makes it easier to find and destroy them with other units. While Mine Drop costs more munitions than normal, the Schwimmwagen can take advantage of its speed to find hot spots as needed, in addition to being able to lay down the mines very quickly. Built From Panzer Elite Headquarters [Expand][Hide] Health 1500 Target Type building Cost 500 Critical Type panel_building Time 61 Hotkey Effects Deploy Pioneers and escalate Battle Phases to call in additional Reinforcements. ?ESeeStructure Panzer Elite Headquartersfor details. Doctrinal Abilities Ability Mark Target Ability Schwimmwagen Mine Drop Ability Incendiary Trap Vehicle Abilities Vehicle Cover [Expand][Hide] Cost Activation always_on Duration _ Target tp_any Recharge 0 Hotkey Effects $0 no key ?ESeeAbility Vehicle Coverfor details. Retrieved from http //coh-stats.com/Vehicle Schwimmwagen_Type_128
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25/Nov/2011 probeの解離定数を求める。 I am gonna determine Kd value. 2 units/ml Bc-PLC 2/5 units/ml Bc-PLC 2/5*5 units/ml Bc-PLC 2/5*5*5 units/ml Bc-PLC 2/5*5*5*5 units/ml Bc-PLC 2/5*5*5*5*5 units/ml Bc-PLC 2/5*5*5*5*5*5 units/ml Bc-PLC 2/5*5*5*5*5*5*5 units/ml Bc-PLC 0 units/ml Bc-PLC At the same time, I am gonna determine real amount of DAG level by brigh and dyer method. 18/Nov/2011 SMの量におけるD609の影響をMDCK細胞で調べる。 Effect of D609 on SM level in MDCK cells 1)ctrl 2)SMase 3)3hour D609 incubation 4)6hour D609 incubation 5)20hour D609 incubation mCherry-lysenin conc is 50microg/ml. 1 30 3hour 4 30 6hour 6 00 20hour SMase 1 units/ml 10 min. After that, fixed. What I have to prepare is that HBSS and 3 % PFA Effect of DGAT inhibitor on DAG level in MDCK cells. 1microM A922500 was used for this experiment. 2pm Add A922500 start incubation 4pm Add A922500 start incubation 5pm DAG probe incubation. こんなかんじで 17/Nov/2011 Rat red blood cell experiment, At first, I did the experiment as follows 1)1U/ml Pc-PLC + 200times annexin V + 9*108 cell/ml result hemolysis within 4 min 2)0.1U/ml Pc-PLC + 40 times annexin V + 9*108 cell/ml result hemolysis within 5 min In this time, protocol was wrong. 3)real experiment 0.1U/ml Pc-PLC + 40 times annexin V + 9*108 cell/ml I get annexin V fluorescence but it is not for living RBL cells. At first, RBC was subjected to hemolysis, become ghost, and then become fluorescence. 4)0.1U/ml Bc-PLC + 40 times annexin V + 9*108 cell/ml RBC rarely give hemolysis and no fluorescence. And I tryed the experiment whether Bc-PLC makes DAG on the plasma membrane in RBC in a Bc-PLC conc. dependent manner. 1) 800 microl RBC (9*108 cell/ml) 2) Add DAG probe 24 microl here And 3)2microl 100 units/ml Bc-PLC was added to the 8 microl HBSS. 4)2microl of this solution was added to next tube, which has 8 microl HBSS. 5)this step is done one after another. this means that 5 time solution is gonna prepare. Next in each tube, 75 microl RBC solution was added and is incubated for 10 min @r.t. after that, fixed 6% (final 3%), and incubated for 15min. and spin down. こんな感じでやりましたよーん。 30/Oct/2011 D609の濃度 50microg/ml Offered as 5 mg (sc-201403) and 25 mg (sc-201403A) sizes Synonym O-Tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt CAS Number 83373-60-8 Molecular Weight 266.5 Molecular Formula C11H15OS2K Purity 98% Physical Appearance Off-white solid Solubility Soluble in water (25 mg/ml) I used D609 with the concentration of 188microM 以下のものをやってみる。 Other experiments 1. living MDCK cells with alpha PMA and PMA 2. DAG probe calibration with Bc-PLC Bc-PLC 20 units/ml Bc-PLC 10 units/ml Bc-PLC 5units/ml Bc-PLC 0units/ml D609 3. fixed MDCK cells with SMase noSMase SMase SMase+D609 SMase+BcPLC 4. living MDCK cells with SMase + Bc-PLC 5. SM amount with SMase or nojirimycin 6. DAG amount with SMase or nojirimycin 111016 もう一度最後の実験 以下の実験を2回繰り返す。 それぞれについて10個の細胞をとる。 それでanalyzeして終わり。 Thinking about biological significance, I am gonna try this experiment as follows. 1. noSMase 2. SMase 3. SMase+D609 4. SMase+BcPLC 16/June/2011 Now I started to write the papers. But I have several experiments I have to do and I want to do. 1. test if the DAG which exist already on the membrane is abolished by incubated with D609. 2. I am gonna take beautiful data of cholesterol, DMS and NB-DNJ. SMS2がPC-PLCであるか否か?とういうことに関係するのですが、 D609処理した細胞で、ライセニンで染まるかどうかという実験を過去にラボでやった人はいらっしゃいますか? あと、SMS1およびSMS2が欠損したMEF-ZS2とSMS1もしくは、SMS2が発現させた細胞では、ライセニンで染めるとどのように違いがでますか? こちらについては、やっているのではないかと思い聴いて見ました。 And then I want to get the PC-PLC antibody. 20May2011 I am gonna do the experiment. I don t miss to write but in the previous experiment. UV was irradiated to MDCK cells for 1 min. 30 min, 1hour 2hout later, the cells were fixed and observed. 30min there is 30% cells in which DAG probe was translocated. But not so obvious. So I am gonna do 10 min incubation after UVB irradiation. Because some papers says that 10 min is more DAG. So, 1. UVB 10min 2. UVB D609 10 min 3. No UVB And then the previous experiment that when MDCK was fixed, DAG probe localized at the plasma membrane incubated with 12.5 Bc-PLC 10 min. So, I need to play around the concentration of Bc-PLC. 4. Bc-PLC 0 microunit/ml 5. Bc-PLC 0.1 microunit/ml 6. Bc-PLC 0.5 microunit/ml 7. Bc-PLC 1 microunit/ml 8. Bc-PLC 5 microunit/ml 9. Bc-PLC 12.5 microunit/ml And then, U73122 doesn t abolish ATP-induced DAG on the outer leaflet of plasma membrane. So, I suspect PAP-induced DAG. I am gonna use the propranolol. 10. ctrl 11. D609 12. D609+ATP 13. D609+ATP+propranolol At the same time, I am gonna use the MDCK cells expressing mGluR5 and bath application of glutamate. 14. ctrl 15. glutamate 06May2011 I gonna do the experiment about DAG flip in fixed cells. A. EYFP-C1AB and DsRed B. EYFP-C1AB and DsRed with 10mM MbCD + 12.5unit/ml BcPLC C. EYFP-C1AB and DsRed with 20microM Fumonisin B1 + 12.5unit/ml BcPLC D. EYFP-C1AB and DsRed with 50nM ISP1 + 12.5unit/ml BcPLC E. EYFP-C1AB and DsRed with 6 microM DMS + 12.5unit/ml BcPLC F. EYFP-C1AB and DsRed with 100 microM NB-DNJ + 12.5unit/ml BcPLC G. EYFP-C1AB and DsRed with Bc-PLC as ctrl H. EYFP-C1AB and DsRed with PMA 28Apr2011 How to irradiate UVB (290-320nm) 1. reduce the volume of medium, DMEM 100 microl. Go to program 2. Set 10 mJoules/cm2 for 1min 3. replace the medium with appropriate media. That s it. 27Apr2011 Now Francoise kindly decide the condition of UVB irradiation. So I am doing the following experiments under the condition. I will observe DG probe and PKCdelta antibody accumulation. 1. Ctrl(negative) Just MDCK cells. 2. Ctrl(positive) PMA 3. UBV and leave 30 min 4. UBV and leave 1 hour 5. UBV and leave 3 hour 7. UBV and leave 30 min with D609 (D609 nothing after UVB) 8. UBV and leave 1 hour with D609 (D609 nothing after UVB) 9. UBV and leave 3 hour with D609 (D609 nothing after UVB) 10. UBV and leave 3 hour with D609 (D609 still incubation after UVB) 11. no D609 12. D609+100microM ATP 13. D609+100microM ATP+1microM U73122 22Apr2011 Thinking about the experiment I have to do. 1. I can get PKCgamma antibody within next week. So I gonna do the following experiment. A. EYFP-C1AB, PKCgamma antibody, DsRed. B. EYFP-C1AB, PKCgamma antibody, DsRed with SMase. C. EYFP-C1AB, PKCgamma antibody, DsRed with SMase and D609. 2. check whether DAG flop is inhbited in the presence of U73122. Actually when I did this expeirment previously using 100 microM U73122, unexpectedly more bright fluorescence comes up. Maybe some artifact. So I gonna reduce the concentration of U73122. A. 100 microM ATP B. 100 microM ATP, 10 microM U73122 C. 10 microM ATP D. 10 microM ATP, 10 microM U73122 3. I am gonna observe the effect of MbCD, FumonisinB1, ISP1, DMS and NB-DNJ in fixed cells. A. EYFP-C1AB and DsRed B. EYFP-C1AB and DsRed with 10mM MbCD + 12.5unit/ml BcPLC C. EYFP-C1AB and DsRed with 10microM(?)Fumonisin B1 + 12.5unit/ml BcPLC D. EYFP-C1AB and DsRed with 50nM ISP1 + 12.5unit/ml BcPLC E. EYFP-C1AB and DsRed with 6 microM DMS + 12.5unit/ml BcPLC F. EYFP-C1AB and DsRed with 100 microM NB-DNJ + 12.5unit/ml BcPLC 07Apr2011 Thinking about discussion with Francoise. 1. I am giving Francoise MDCK cells. 2. I gonna show the paper I am preparing now. 3. I gonna give her D609, rottelin Reiko has. 4. I gonna give her the paper relationship with D609 and apoptosis, with rottelin and apoptosis, and MDCK and apoptosis, SMase and apoptosis. 25/Mar/2011 Thinking about biological significance, I am gonna try this experiment as follows. 1. noSMase 2. noSMase+U73122 3. noSMase+D609 4. SMase 5. SMase+U73122 6. SMase+D609 7. SMase+BcPLC 8. SMase+BcPLC+U73122 Express EYFP-C1AB, DsRed and PLCepsilon-HA Express EYFP-C1AB, DsRed and PLCepsilon-HA(dominega) Express EYFP-C1AB, DsRed and PLCalpha-HA Express EYFP-C1AB, DsRed and PLCalpha-HA(dominega) 24/Mar/2011 And then, I found another contraversy. In IPS1 experiment, flip doesn t occure even though without SM. I need to do same experiment with SMase. 22/Mar/2011 I am gonna do the following experiments from the data I took on 15/Mar/2011. At first, 10 microM U73122 experiment doesn t work. So I am gonna do increase its concentration or decreases in ATP concentration. This experiment is very critical to say that the EYFP-C1AB fluorescence is caused by DAG drived from PI-PLC. 1. 100 microM ATP 2. 100 microM ATP, 100 microM U73122 3. 100 microM ATP, 100 microM U73343 4. 10 microM ATP 5. 10 microM ATP, 100 microM U73122 6. 10 microM ATP, 100 microM U73343 Another point I have to mention is that 24 hour incubation with U73122 did not give the difference in outer DAG signal compared to ctrl. This means that PI-PLC doesn t contribute to outer DAG. But, I can see PI-PLC dependent outer DAG sigal. So I want to make clear the signal from PI-PLC caused by ATP addition is transient or sustained. I guess this signal is transient. I am gonna do 10min, 30min, 1 hour, 2 hour. In some case I am gonna remove the ATP in the middle of reaction. 15/Mar/2011 I am gonna take reproduciblity. 1. MDCK Cameleon with ATP no D609 2. MDCK Cameleon with ATP with D609 3. MDCK control no D609 4. MDCK D609 only 5. MDCK D609 ATP 6. MDCK D609 ATP U73122 7. MDCK D609 ATP U73343 8. MDCK D609 ATP R59949 9. MDCK D609 ATP SMase 10. MDCK D609 PMA 11. MDCK D609 DiC8 12. MDCK C1AB inner no D609 13. MDCK C1AB inner no D609 ATP 14. MDCK C1AB inner D609 15. MDCK C1AB inner D609 ATP 16. MDCK mGluR no D609 17. MDCK mGluR D609 18. MDCK mGluR D609 Result Patially success. I got good result about that in the presence of D609 and ATP the DAG signal was detected, but no signal in the presence of D609 only. Of course, no D609 gives very bright fluorescence. But disappointed point is that U73112 did not inhibit the fluorescence which is caused by addition of ATP. Interpretation of it is due to week effect of U73122. I need to increase in concentration of U73122 or decrease of concentration of ATP. And then, the points I need to condider is that in the presence of U73122, which is 24 hours incubation, DAG signal did not decreases. But transient increase caused by inner DAG caused by ATP through PI-PLC is detected. How do I come to term with these. 09/Mar/2011 The idea to observe DAG flop. 1. no D609 2. ctrl 3. ATP 10 min 4. ATP U73122 10 min 5. ATP R59949 10 min 6. ATP SMase 10 min 7. PMA 10 min 8. DiC8 10 min 9. Inner C1AB ATP 10.Inner C1AB ATP 1. About 9 and 10, C1AB is transfected one day before. 2. 2-10 is incubated with D609 for 6 hours. 3. 1067microl(97times11)HBSS is prepared 4. 33microl C1AB is put, means total 1100microl 5. 100microl pick up. Number1 6. 4microl D609 is put, total 1004 microl 7. Divide in half 8. first half, 5 microl ATP is added. 9. 100 microl pick up. Number2. 10. From remaining 400microl, each 100 microl is taken, add 1 microl PMA(10microM) and 1 microl DiC8 (100microM).Number 7 and Number 8. 11. About second half, 4microl ATP is added. 12. 100microl is picked up, Number3. 13. 1 microl U723122(15microM), R59949(10microM) or 2microl SMase(1unit) is added to each 100 microl, Number 4,Number5 and Number 6. Result In some part it is works well. But I missed to enter D609 in the cell which was supposed to be in the presence of U73122. So I need to do again about U73122. And My concern is C1AB probe expressed into the cells localized to the membrane in the presence of only D609. I need to chech this is really real or not. And I would be better to confirm that P2Y2R stay on the surface on the plasma membrane, by doing measure increase in calcium concentration. c 04/Mar/2011 I prepared WR19L cells, lymphocyte. I am gonna do the following experiments. I am gonna add 10 microM PMA, 100 microM DiC8, 12.5 units/ml Bc-PLC, 10 microM C6-Cer mixed with EYFP-C1AB probe. Experimental procedure WR19Lcells were spind down and collected. Cells were in total 815 microl(163microl*5 in each)HBSS solution. Add 25microl 3.8mg/ml EYFP-C1AB. 2microl 1mM PMA, 1mM alphaPMA, 100mM DiC8, 8.5microl of 12.5unit/ml Bc-PLC are put in the 1.5 ml tubes in advace. 168microl solution containing EYFP-C1AB is added in each tube and incubate 10 min. 30micro 20% PFA is added and fix for 10 min. After that, spine down cells and remove supernatant. Add 50mM NH4Cl for 5min. Spine down and suck up supernatant. Add 100microl HBSS. Result I could do it! I can see brighter fluorescence in PMA than that in control.About DiC8, It seems that there is no difference compared to control.About Bc-PLC, I could see very bright fluorescence in the cells which is aggregated, indicating that DAG is important for membrane fusion. 04/Mar/2011 I prepared WR19L cells, lymphocyte. I am gonna do the following experiments. I am gonna add 10 microM PMA, 100 microM DiC8, 12.5 units/ml Bc-PLC, 10 microM C6-Cer mixed with EYFP-C1AB probe. Experimental procedure WR19Lcells were spind down and collected. Cells were in total 815 microl(163microl*5 in each)HBSS solution. Add 25microl 3.8mg/ml EYFP-C1AB. 2microl 1mM PMA, 1mM alphaPMA, 100mM DiC8, 8.5microl of 12.5unit/ml Bc-PLC are put in the 1.5 ml tubes in advace. 168microl solution containing EYFP-C1AB is added in each tube and incubate 10 min. 30micro 20% PFA is added and fix for 10 min. After that, spine down cells and remove supernatant. Add 50mM NH4Cl for 5min. Spine down and suck up supernatant. Add 100microl HBSS. 18/Feb/2011 I am gonna follow Malhotra s procedure. I prepared the liposomes as follows. 1. POPC only=60 2. POPC/PS=60/20 3. POPC/GM1=60/20 These liposome includes 2mol% Rhodamine-PE 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. PS(0.04microl)avanti 100721 2.3mM 0.45 microl 3. GM1(0.04micromol) sigma G7641 1mM 1.128 microl 4. Phodamin-PE(4nmol) Avanti 0.15 mM 28 microl 1. I diluted these liposomes in 666microl, 888microl, 888microl in each. This means that 300 microM liposomes. 2. I am gonna pick up 100 microl in each. 3. EYFPC1AB domain is 1 mg/ml I am gonna add 1microl into 100 microl. This means that 10 microg/ml. 4. About DiC8, I have 1M DiC8 now (DMSO). I am gonna dilute this solution 200 times(PBS(-)), this means 5mM. Add 1microl into 100 microl. I want to prepare as follow. A. POPC B. POPC+probe C. POPC+probe+DiC8 D. POPC/PS E. POPC/PS+probe F. POPC/PS+probe+DiC8 G. POPC/GM1 H. POPC/GM1+probe I. POPC/GM1+probe+DiC8 5. FRET mesurement.30 min incubation. excitation 488 nm emission 500-650nm 6. Flotation assay (I can only use 4 samples for ultracentrifugation) B. POPC+probe C. POPC+probe+DiC8 H. POPC/GM1+probe I. POPC/GM1+probe+DiC8 7. Normalize with fluorescence of liposomes. 8. Apply sample solution to SDS-Page. 9. Stain with GFP antiboly. 06/Feb/2011 I couldn t make liposomes previously in 03/Feb/2011. However, I leave these lipid films for 1 week in the room condition. This might cause some bad effect on these membranes. So, I gonna make liposome again with same condition with littele modification. Because previous study use fluorescence PE was used 2mol%. I am gonna increase the volume of Rhodamine-PE from 0.5 to 2 mol%. So, 1. POPC only 2. POPC/DAG=60/6 3. POPC/DAG/GM1=60/6/20 4. POPC/DAG/PS=60/6/20 5. POPC/PS=60/20 6. POPC/GM1=60/20 Phodamine-PE is inluded all 2 mol%. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. PODG(0.033micromol) avanti 800815C 3.4 mM 9.8 microl 3. PS(0.04microl)avanti 100721 2.3mM 0.45 microl 4. GM1(0.04micromol) sigma G7641 1mM 1.128 microl 5. Phodamin-PE(4nmol) Avanti 0.15 mM 28 microl So then, I am gonna prepare following. 1. Take 102 microl POPC and 196 microl PE. 2. Mix and take 42.6 microl each in 6 tubes. 3. 9.8microl DAG is added 2,3,4 tubes. 4. 1.1microl GM1 is added 3,6 tubes. 5. 0.5microl PS is added 4,5 tubes. result I try sonication extensivly. But didn t make liposomes containing DAG even though biophysical journal paper shows that they could make liposomes. 03/Feb/2011 Lipid filmes prepared in 20/Jan/2011 was voltexed. But 5,6 didn t make liposomes. 2,3,4 did but rim of glass tube have fluorescence lipids, I am afraid of phase separation of DAG on the glass tube. Only 1 made liposomes well. 20/Jan/2011 I did the experiment of 09/Jan/2011 But this experiment didn t work well. Even positive control didn t work well, which is same result of ctrl. Toshi-sensei said that the distance between fluorescent PE and EYFP-C1AB doesn t become close enough. So I am gonna give up this experiment. I am gonna do following expeiment. I will prepare following liposomes. 1. POPC only 2. POPC/DAG=60/6 3. POPC/DAG/GM1=60/6/20 4. POPC/DAG/PS=60/6/20 5. POPC/PS=60/20 6. POPC/GM1=60/20 Phodamine-PE is inluded all 0.5 mol%. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. PODG(0.033micromol) avanti 800815C 3.4 mM 9.8 microl 3. PS(0.04microl)avanti 100721 2.3mM 0.45 microl 4. GM1(0.04micromol) sigma G7641 1mM 1.128 microl 5. Phodamin-PE(1nmol) Avanti 0.15 mM 7 microl So then, I am gonna prepare following. 1. Take 102 microl POPC and 49 microl PE. 2. Mix and take 21.6 microl each in 6 tubes. 3. 9.8microl DAG is added 2,3,4 tubes. 4. 1.1microl GM1 is added 3,6 tubes. 5. 0.5microl PS is added 4,5 tubes. 09/Jan/2011 I read a paper in which they made liposomes. I will modify a little bit. And I am gonna detect FRET signal. ex 488 nm, em 560 nm. 1.POPC/BODIPYPE(530/560)=60/0.6 2.POPC only (as ctrl) 3.POPC/PS/BODIPYPE(530/560)=60/6/0.6 (positive ctrl) 4.POPC/GM1/BODIPYPE(530/560)=60/6/0.6 5.POPC/GM1=60/6 (as ctrl) Each is divided into two samples. One is for ctrl. The other is for Bc-PLC addition. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. BODIPYPE(530/560)(0.004micromol) Molecular probe 1 mM 2microl 3. PS(0.02microl)avanti 100721 2.3mM 0.245 microl 4. GM1(0.02micromol) sigma G7641 1mM 0.564 microl 03/Jan/2011 I did the condition of 02/Dec. But this lipid mixture did not make liposomes more than previous condition. So Next thinking is that remove cholesterol. That menas that POPC/SM/DAG=60 20 10 and so on. And I did the experiment in which I see the effect of C1AB on cell growth. MDCK and HEK cells. I cultured HEK cells as following condition. cell concentration is 1.62*106 cells/ml. 10microl solution was added to 500microl medium. After that 10microl 3.8mg/ml C1AB was added. Ctrl is only 10microl 200mM imidazole buffer solution. About HEK cells, 3.0*105 cells/ml was prepared. 30microl this solution was added to 500microl DMEM solution. After that, 25microl 3.8mg/ml C1AB solution was added. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. brainSM(0.066micromol) avanti 860062C 31.4mM 2.1 microl 3. chol(0.2micromol) unknown 10.2mM 19.6microl 4. PODG(0.033, 0.066micromol) avanti 800815C 3.4 mM 9.8, 19.6 microl So, I made such liposomes. 1. POPC=60 2. POPC/DAG=60/5 3. POPC/DAG=60/10 4. POPC/SM=60/20 5. POPC/SM/DAG=60/20/10 6. POPC/SM/DAG=60/20/20 02/Dec/2010 It turns out that these lipid mixture I prepared in 30/Dec did not form liposomes. So, I asked Toshi-sensei how to make liposomes well. うえだくん、 コレステロールを増やせばうまくいくかも知れません。 POPC/SM/chol/DAG=60 20 60 10とか。 試してみてもらえますか? 小林 So I made liposome as follows. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. brainSM(0.066micromol) avanti 860062C 31.4mM 2.1 microl 3. chol(0.2micromol) unknown 10.2mM 19.6microl 4. PODG(0.033, 0.066micromol) avanti 800815C 3.4 mM 9.8, 19.6 microl So, I made such liposomes. 1. POPC/SM/chol=60/20/60 2. POPC/SM/chol/DAG=60/20/60/10 3. POPC/SM/chol/DAG=60/20/60/20 30/Dec/2010 I prepared liposomes newly. SM was included because SM is important for distribution of DAG on the membrane. And then, I mimicked the outer leaflet of the plasma membrane. So, I used as follows 1. POPC(0.6micromol) avanti 13.7mM 43.8microl 2. brainSM(0.2micromol) avanti 860062C 31.4mM 6.3 microl 3. chol(0.2micromol) unknown 10.2mM 19.6microl 4. PODG(0.1, 0.2, 0.3,0.4 micromol) avanti 800815C 3.4 mM 29.4, 58.8,88.2,117microl So, I made such liposomes. 1. POPC/SM/chol=60/20/20 2. POPC/SM/chol/DAG=60/20/20/10 3. POPC/SM/chol/DAG=60/20/20/20 4. POPC/SM/chol/DAG=60/20/20/30 5. POPC/SM/chol/DAG=60/20/20/40 28/Dec/2010 EYFP-C1AB 1.77 mg/ml now (M.W=38,000) I took 3 microl this means 1.77 mg*3microl/1000microl=5.31 microg. So 5.31/38000=0.0001397micromol=0.1397nmol. 1 cell have 1 fmol DAG. glass base dish maybe has 10000 cells/1 well. So 1 fmol*10000=10pmol DAG. but outer DAG is less assume 1%. So 100fmol DAG. liposome 19mM DOPC 52.6microl this means 19 mmol/1000000microl*52.6microl=1micromol. 3.2mM DODG 12.5microl this means 3.2mmol/1000000microl*12.5microl=40nmol. These two are mixed, so 4%mol DAG. suspended in 200microl HBSS. 100microl picked up, means 500nmol PC 20 nmol DAG. vesicle contains two side, so 250nmol PC 10nmol DAG I did liposome experiment, but result is mysterious. In the presence of DAG in PC liposome, fluorescence light is brighter than that in the presence of only PC liposome. I think about this. Basically, I don t know what happen, but there is some hint. 1. cells were ripped off when both liposome was added, this means that PC works as detergent because I add a lot of liposome, 500 nmPC. So I need to reduce the amount of PC liposome. 2. In this experiment, I add 4mol% DAG in PC liposome. But other experiment, for example SM-lysenin, give 20mol% DM into PC liposome. So I can add more DAG, maybe at least 10%, possbly 20%-40%. As you know, If DAG included more, this membrane doesn t make a liposome. So I need to take care about it. Next time, I am goning to prepare the liposome, リポソームですが、1mMを1ml位用意して、 蛋白の濃度にもよると思いますが、 こちらの実験では、GFP-Lysの場合、 いつも、50倍希釈で染色しているので、 (1マイクロ/50マイクロPBS) 1マイクロに、50マイクロのリポソームを混ぜて、 37度で30分してから、それで染色していました。 1mMはかなり濃い濃度なので、タンパク質はすべて 結合すると考えています。 さっき探していた論文ですが、(準備中) For the preabsorption experiment, Dronpa-θ-D4 or Dronpa-NT-Lys was incubated with MLVs at 37°C for 30 min prior to apply them to HeLa cells. としか書いてありませんでした。 これで良いですか? 牧野 Reply Forward Reply |Ueda Yoshibumi to Asami show details Dec 17 (7 days ago) リポソームを作るとき、はじめに、クロロホルムに溶けた脂質を遠沈管の中に加えて、 窒素で飛ばしてからPBSなどを加えてベシクルにするんだよね? だいたい、クロロホルムに溶けた1mM脂質(例えばDOPC)を何μl位遠沈管に入れて乾かしていますか? それは脂質のストック濃度によると思います。 10mMの脂質ストックなら100ulだし、20mMなら50です。 それで乾いてから1mlのPBSをいれて、 最終濃度1mMリポソームが 1mlできるということです。 Show quoted text - Reply Forward Reply |Ueda Yoshibumi to Asami show details Dec 17 (7 days ago) 1mMだったら脂質を1ml入れているっていうことだね。 かなり多い量を加えているんだね! なるほど、わかりました。 Reply |Asami Makino to Ueda show details Dec 17 (7 days ago) でも、結局リポソームは50マイクロLとか使わないから、 1mlも作る必要ないと思う。 200マイクロでも作れば充分なんじゃないかな。 ただ、こちらにある脂質のストックはほとんど10mM以上あるので たくさんある脂質は少量はかり取るのが大変だから、 たくさん使ってもいいと思います。 Original Message ----- From Ueda Yoshibumi To Asami Makino Show quoted text - Sent Friday, December 17, 2010 3 31 PM Subject Re MLV なるほど、了解しました! So, I prepared vesicles. I use 10microg/ml YFP-C1AB to stain the cells. YFP-C1AB M.W. 38kDa=38,000 This means 0.000263micromol/ml I add 100microl, So total amount is 0.02nmol. I want to include DAG into the vescle And then, 1 cells has 1 fmol DAG. glass based dishes is maybe 10 pmol DAG.