Assay of B16F10 intracellular tyros

Assay of B16F10 intracellular tyrosinase activity Cellular tyrosinase activity was determined as described previously [34] with slight modifications. Briefly, the cells
were treated with α-MSH (100 nM) for 24 h, and then intracellular tyrosinase activity was measured after treatment with various concentrations of M. grandiflora L.
flower extract (final concentration 10, 15, 20%; v/v) or arbutin (2.0 mM) for 24 h. After these treatments, the cells were washed twice with phosphate-buffered saline
and homogenized with 50 mM PBS (pH 7.5) buffer containing 1.0 % Triton X-100 and 0.1 mM PMSF. Intracellular tyrosinase activity was monitored as follows: Cell extracts (100 μL) were mixed with freshly prepared L-DOPA solution (0.1% in phosphate-buffered saline)
and incubated at 37°C. The absorbance at 490 nm was measured with microplate reader Gen 5TM (BIO-TEK Instrument,Bermont, USA) to monitor the production of dopachrome, corrected for auto-oxidation of L-DOPA.

結果 (繁體中文) 2:

B16F10胞內酪氨酸酶活性的細胞的酪氨酸酶活性的測定如前[34]稍作修改來確定。簡言之，將細胞
分別用α-MSH（100納米）24小時處理，然後細胞內酪氨酸酶活性，用各種濃度的分枝玉蘭L.治療後測量
花提取物（最終濃度10，15，20％;體積/ v）或熊果苷（2.0毫摩爾）24小時。這些處理後，將細胞用磷酸鹽緩衝鹽水洗滌兩次
，並勻化用50mM的PBS（pH7.5）中含有1.0％的Triton X-100和0.1mM PMSF緩衝液中。細胞內的酪氨酸酶的活性監測如下：細胞提取（100μL）中混合新鮮製備的L-DOPA溶液（在磷酸鹽緩衝鹽水0.1％）
並在37℃。490nm處的吸光度的測定使用酶標儀根5TM（BIO-TEK儀器，Bermont，美國），以監控生產多巴色素的，用於自動氧化的L-DOPA的校正。

結果 (繁體中文) 3:

確定先前所描述的[ 34 ]稍作修改了B16F10細胞內酪氨酸酶的活性細胞酪氨酸酶活性測定。簡單的細胞，
與αMSH（100 nm）處理24 h，然後細胞內酪氨酸酶的活性與不同濃度的廣玉蘭屬
花提取物治療後量測（終濃度為10，15，20%；V / V）或熊果苷（2毫米）24 h處理後，細胞用磷酸鹽緩衝鹽水
和均質50毫米的PBS洗兩次（pH 7.5）含有1%的Triton X-100和0.1毫米PMSF緩衝。細胞內酪氨酸酶的活性監測如下：細胞提取物（100μL）混合新鮮製備的左旋多巴溶液（0.1%的磷酸鹽緩衝生理鹽水）
並分別在37°C.在490 nm處的吸光度與酶標儀量測根5tm（寶特儀器，柏蒙特，美國）監測多巴色素的生產，對左旋多巴的自動氧化校正。