Dermal fibroblasts were seeded on 6

Dermal fibroblasts were seeded on 60-mm plates and incubated for 1 day. And, cells were irradiated with UVA 6 J/cm2 and then incubated overnight in Iscove’s modified Dulbecco’s medium containing 10% FBS and RG NG or RG WE (0, 100, 250, or 500 mg/ml). The conditioned media were collected using an Amicon Ultra-4 centrifugal filter (Millipore, USA). The proteins in the media were concentrated by centrifugation (13,000 g for 3 h at 4C). The proteins were separated on 10% zymogram gel (Invitrogen, USA), and the gels were renatured in 2.5% Triton X-100 and then were developed in buffer with 50 mM Tris-HCl, pH 7.6, 1 mM CaCl2, 0.2 M NaCl overnight at 37C. The gels were stained with Coomassie blue (Brilliant blue G solution; Sigma, USA) and destained in buffer with 10% acetic acid and MeOH. Gelatinolytic activity, i.e., unstained areas on gel, was quantified by using Luminograph II and CSAnalyzer4 program (Atto, Japan). Zymography was performed in triplicates for each sample.

結果 (繁體中文) 1:

真皮成纖維細胞接種在60毫米的板，並溫育1天。並且，將細胞用UVA 6焦耳/平方厘米照射，然後在Iscove氏含有修飾的Dulbecco培養基中孵育過夜10％FBS和RG NG或RG WE（0，100，250，或500毫克/毫升）。使用Amicon超離心4濾器（Millipore，USA）收集條件培養基。在介質中的蛋白通過離心（在4℃13000克3小時）濃縮。將蛋白質分離在10％酶譜凝膠（Invitrogen，USA），將凝膠中進行复性2.5％的Triton X-100，然後在緩衝液中開發的用50mM的Tris-HCl，pH值7.6，1mM的氯化鈣，0.2M NaCl的隔夜在37℃。凝膠用考馬斯藍（亮藍G溶液; Sigma，美國）染色，並在緩衝液中，用10％乙酸和MeOH脫色。溶明膠活性，即對未染色凝膠領域，通過使用Luminograph II和CSAnalyzer4程序（阿托，日本）來定量。酶譜法一式三份對各樣品進行。

結果 (繁體中文) 2:

你說的是這個星期六，是嗎？你什麼時間需要離開你目前的家？

結果 (繁體中文) 3:

真皮成纖維細胞接種於60mm培養板上，孵育1d。用UVA 6 J/cm~2照射細胞，然後在Iscove改良的含有10%FBS和RG-NG或RG-WE（0、100、250或500 mg/ml）的Dulbecco培養基中培養過夜。使用Amicon Ultra-4離心篩檢程式（美國密理博）收集條件培養基。用離心法濃縮培養基中的蛋白質（13000g，4C下3h）。在10%酶譜凝膠（美國因維特羅根）上分離蛋白質，在2.5%Triton X-100中複性，然後在50 mM Tris HCl、pH 7.6、1 mM CaCl2、0.2 mNaCl緩衝液中於37℃過夜，用考馬斯藍（亮藍G溶液；Sigma，（美國）並用10%醋酸和甲醇緩衝。凝膠溶解活性，即凝膠上的未染色區域，通過使用發光儀II和CSAnalyzer4程式（日本阿托）進行量化。對每個樣本進行三次酶譜分析。