A method for the direct measurement of intracellular nitric oxide (NO) production stimulated by penicillin G (PG) in cultured hippocampal neurons with diaminoanthraquinone (DAA) using laser scanning confocal microscopy (LSCM) was developed. Intracellular DAA fluorescence could specifically represent NO production based on two facts: (1) 3-morpholinosydnonimine, a NO donor, could dose-dependently increase DAA fluorescence; and (2) haemoglobin, a NO scavenger, could inhibit the increase of DAA fluorescence. The PG dose-dependently increasd the intercellular level of glutamate (Glu, 5 min after stimulation) and the intracellular NO production (30 min throughout stimulation). The increase of NO production could be reversed by Nw-nitro-L-arginine (a NO synthase inhibitor), and also by D(-)2-amino-5-phosphonovaleric acid, a subtype of Glu receptor antagonist. These results revealed that DAA could be used to indicate real-time and kinetic intracellular NO production of hippocampal neurons with higher sensitivity, specificity and accuracy.

A novel method based on flow cytometry (FCM), which can count the number of detected cells, has been developed for the evaluation of cellular proliferation and cytotoxicity in vitro. It provides a tool that directly counts cell number without being influenced by the metabolic state of the cells, discriminates target cells from effector cells in cell-mediated cytotoxicity assay, and with less treatment step and free radioactivity. In this paper, we have prepared the PG cells (a highly metastatic human lung cancer cell line) and peripheral blood lymphocytes (PBL) with various concentrations and ratios of concentration to validate the method. The results were compared with MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) assay and the regression analysis results showed that this method worked very well. We have also used this method to evaluate mitogeninduced proliferation and cytotoxicity. The results indicated that this method might yield high sensitivity and reliability.

Nitric oxide (NO) and calcium ions (Ca2) play critical role as molecular mediator in many physiological processes. However, their low concentration and instability in specimen make them difficult to be detected directly in neurons. We developed a method for imaging nitric oxide and calcium ions using Laser Scanning Confocal Microscopy (LSCM). Cultured hippocampal neuron is dyed and observed under Zeiss LSM 510 laser scanning confocal microscope. Excited by laser the emission light from the labeled nitric oxide and calcium ions in the neutron are detected. In this way, the nitric oxide and calcium ions are imaged and their intracellular kinetic change is monitored. Furthermore, image processing and visualization techniques are employed to help analyze the image data.

建立一个用于体外研究特定流动剪切力作用下白细胞和内皮细胞动态相互作用的方法。利用建立的平板流动小室系统可在体外产生特定的流动剪切力。将培养的人脐静脉内皮细胞装入平板流动小室后，以0.71dynes/cm2的流动剪切力把含有吖啶橙染色的白细胞的灌流液导入流动小室，由此产生了白细胞和内皮细胞的动态粘附过程。整个粘附过程通过OlympusIX70倒置荧光显微系统观察，同时通过CCD摄象头录像。然后用图象采集卡将录像采集为数字图象并保存。利用针对实验设计的图象处理和分析方法，对采集的数字图象进行处理和测量，可以得到粘附白细胞的个数和滚动白细胞的速度。通过研究内毒素脂多糖（LPS）对内皮细胞粘附功能的促进及地塞米松（DXM）对该刺激的抑制作用来验证。对于用内毒素脂多糖（LPS）处理的内皮细胞，固定粘附和慢速滚动的白细胞的个数比对照组分别显著增加了23.7倍和4.1倍，同时白细胞在粘附作用过程中慢速滚动和快速滚动的速度比对照组明显降低了25.6%和26。1%而对于脂多糖和地塞米松（DXM）处理过的内皮细胞，上述内毒素引起的影响被显著抑制了。该方法可以用于研究不同的化学和物理刺激对内皮细胞功能的影响机制，及用来评价各类抑制内皮细胞粘附功能的药物。