本工作旨在探讨贴壁培养细胞台盼蓝拒染试验的方法, 为其合理应用提出建议。用正常培养及NaN3作致死处理的人视网膜色素上皮细胞株-19, 进行原位贴壁细胞和培养体系上悬液中细胞的台盼蓝拒染实验, 并比较其原位法与计数板法所测活细胞率。与计数板法相似, 随染液浓度增高或染色时间延长, 原位台盼蓝拒染实验的活细胞率检测值逐渐增加。培养体系上悬液中脱落细胞的存活率显著低于贴壁的细胞, NaN3处理使脱落细胞增加。因此, 对于贴壁牢固的培养细胞, 原位台盼蓝染色是检测活细胞率的实用方法。根据细胞从培养表面上消化下来的难易程度和脱落到培养上悬液中的数目多少, 可选用原位染色法或/和计数板法台盼蓝拒染试验, 从而检测贴壁培养细胞的总存活率。

The present work aimed at making an approach to the trypan blue exclusion test (TBET) for cultured adherent cells, and offering suggestions for its adequate application. Human retinal pigment epithelial cell line-19 (ARPE-19) was used in this work. The cells were cultured in growth medium with or without the addition of sodium azide (NaN3). The survival percentage of attached cells was measured by in situ TBET as well as hemacytometer-based one. The latter one was performed for detached cells in the culture supernatant. Similar to the knowledge of hemacytometer-based TBET, measured values of percent cell survival by in situ TBET increased gradually with the increase in either dye concentration or staining time. The survival percentages of detached cells were markedly lower than those of attached cells. The number of detached cells increased extensively owing to NaN3 exposure. Therefore, the in situ TBET is a practical method for measuring the survival percentage of cells attached tightly to the culture substrate. According to the difficulty level of enzymatically detaching cells from culture surfaces and the number of spontaneously detached cells in culture supernatant, the in situ TBET or/and the hemacytometer-based one can be selected to determine the overall survival percentage of cultured adherent cells.