多聚左旋赖氨酸包被玻片的可视化检测方法

分子量15万—30万的多聚左旋赖氨酸(Poly-L-Lysine, PLL)常被用于盖玻片的包被,以促进培养细胞贴壁或切片组织黏附。然而,用市场上劣质的PLL将使组织或细胞不能牢固黏附在包被玻片上,进而导致实验失败。因而,建立实用直观的PLL质量检测方法,对于PLL包被玻片的质量控制至关重要。然而,目前世界上未见相关方法报道。因此,作者本着实用原则建立了一种实验室可视化检测盖玻片表面PLL的方法,这种方法利用化学反应将异硫氰酸荧光素(Fluorescein isothiocyanate, FITC)添加到PLL的侧链氨基(-NH2)上,再利用荧光成像检测附着在玻片表面的FITC-PLL,同时荧光照片也可以通过Fiji软件进行分析,获得定量的检测结果。利用此方法,研究发现利用不同浓度的PLL包被盖玻片后, PLL偶联的FITC荧光强度和PLL浓度成正相关。通过检测市面上的两种商品化PLL,与对照相比,两种商品化PLL附着密度极低,推断利用这两种不合格的PLL包被玻片,将导致切片样本从玻片上脱落。此方法适用于实验室自制或商品化PLL黏附盖玻片质量的可视化检测，也为附着在玻璃或塑料表面的带有...

VISUAL DETECTION OF POLY-L-LYSINE ON THE COVER GLASS

Poly-L-Lysine (PLL) with a molecular weight of 150000—300000 is often used in cell culture and histology to promote cell or tissue adhesion. After treating the cover glass with 0.05%—0.1% PLL solution, PLL adhered to the surface of the cover glass to form a monolayer. The density of the PLL directly affects the adhesion effect of the cover glass. However, because PLL is colorless and transparent, it is difficult to distinguish the PLL density and distribution on the surface of PLL-treated cover glass with the naked eye, so it is crucial to establish a simple and effective PLL detection method. However, there are still no relevant methods reported worldwide. In this study, a laboratory visual and quantitative detection method of PLL was developed. In this method, the fluorescein isothiocyanate (FITC) is added to the side chain amino group (-NH2) of the PLL on the surface of the cover glass in sodium bicarbonate solution, and fluorescence imaging is applied to detect the FITC-PLL. In this study, we firstly coated cover glass of 0.1% PLL of three different brands (S, M and A), and found that only the brand S PLL-coated cover glass still had intact tissue section after several washes with phosphate buffer. Accordingly, we found that the FITC-PLL of brand S on the cover glass was bright and uniform, while both the FITC-PLL of brand M and A were dim (3.4% for M, and 4.5% for A compared with S). Therefore, this method is suitable for the quality control of the homemade or commercial PLL-coated cover glass. Furthermore, the method also provides a reference for the establishment of a visual and quantitative detection method of material with a -NH2 group attached to glass or plastic surface.