- 期刊
A Tetrazolium-Based (MTT) Assay for Measuring Viability of Cultured Porcine Corneal Endothelium
以MTT比色試劑法測量豬眼角膜內皮細胞之存活性
摘要
MTT (3- (4, 5- dimethylthiazol-2-yl) -2, 5- diphenyl tetrazolium bromide)方法由於其方便、迅速、簡單和有效的計量活細胞，近年來在各種生物領域廣為運用。如測定抗癌藥物的化學活性，細胞在各種刺激下的增殖性，藥物對細胞之毒性等等。過去吾人以Dye Exclusion Assay來做藥物對於眼角膜內皮細胞毒性，相當繁瑣及花時間。本研究嘗試以MTT方法來評估培養的單層豬角膜內皮細胞的存活性，並找尋最適當波長的光譜以利Scanning Multiwell ELISA Spectrophotometer在讀取吸光度時能獲得最大範圍。另一方面，本研究嘗試找尋在那一個範圍之內的吸光度能與角膜內皮細胞成有意義的線性相關。 我們利用第三代的培養豬眼角膜內皮細胞，殖入96-well microtitration plate。第一和十二直列為空格(即不加入細胞)，中間10個直列，每列(8個well)以50，100，150，1000，1500，5000，10,000，15,000，20,000和30,000個細胞植入每well，重覆兩個plates。第三天保溫箱培養之後，待細胞沈澱附著於盤底，然後將培養液吸乾行MTT方法測量存活細胞數目。以Phosphate Buffer Solution (PBS)溶解MTT，濃度為0.833 µg/ml。MTT試劑加入4小時之後吸起30µl，加入Dimethyl Sulfoxide (DMSO)以溶解MTT轉變成的formazan結晶。以450，540和630nm三種波長光譜在ELISA Spectrophotometer下讀取吸光度。細胞數目與吸光度的線性關係以SAS-PC統計程式做線性迴歸分析。 結果顯示在所測試的三個波長光譜當中，以540nm能獲得最大範圍的吸光度，從0.1193至0.6183。在所測試的三個波長光譜下，吸光度皆能與50至30,000個角膜內皮細胞呈有意義的線性相關。因此在未來的研究當中，我們將選擇以540nm波長之濾鏡於ELISA Spectrophotometer讀取吸光度。而所得之吸光度若介於0.1193至0.6183都能接受以估計角膜內皮細胞的數目。
關鍵字
無資料並列摘要
A tetrazolium-based (MTT) assay was used to evaluate the viability of cultured porcine monolayer corneal endothelial cells. In this study, we tried to find out the best spectra at which scanning multiwell ELISA spectrophometer can obtain the widest range of absorbance (optical density, O.D.) and the ranges of numbers of corneal endothelial cells in which absorbance is in a significant linear relationship with cell numbers. Among the three tested spectra of 450, 540, and 630nm, the widest range of absorbance was at 540nm, from 0.1193 to 0.6183. At all three tested spectra, numbers of corneal endothelial cells form 50 to 30,000 were in a significant linear relationship with absorbance. Therefore, in the future studies to assay the viability of corneal endothelial cells, we shall choose the spectrum of 540 nm for reading with a scanning multiwell ELISA spectrophotometer, and absorbance (O.D.) from 0.1193 to 0.6183 will be accepted, representing cell numbers from 50 to 30,000.