Dr. BR Ambedkar Center for Biomedical Research, University of Delhi, Delhi, 110007, India.
Anal Biochem. 2019 Jul 15;577:42-44. doi: 10.1016/j.ab.2019.04.014. Epub 2019 Apr 20.
In general, 4 amino-5-methylamino-2',7'-difluorescein diacetate (DAF-FM-DA) dye is used to detect nitric oxide in biological systems through cell imaging. In this study, we have used 96 well plate format to quantify nitric oxide using DAF-FM-DA through a multimode reader (or independently using fluorospectrometer) and could be visualized in a fluorescence microscope. Similar study otherwise will require a high-end instrument. The method has been validated to screen NOS inhibitors in the HEK 293T cell lines over-expressing the NOS isoforms. We observed that the method is very simple to use, adaptive, sensitive and most importantly it saves time. REAGENTS/TOOLS: Ethanol (70% [v/v] in distilled water), Nω-Nitro-l-arginine (l-NAME), 7-Nitro-Indazole (7-NI) (Sigma, St. Louis, MO), HEK 293T cell lines (National Centre for Cell Science (NCCS), Pune, India), DMEM (Himedia laboratories Pvt), Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 atmosphere. Hank's Balanced Salt Solution (HBSS) without Phenol Red of pH 7.4 was prepared with the following composition: NaCl, 8.0g, KCl, 0.4g, CaCl, 0.14g, MgSO⋅7HO, 0.1g, MgCl·6HO, 0.1g, NaHPO·2HO, 0.06g, KHPO, 0.06g, glucose, 1.0g, NaHCO, 0.35g, HO, to 1000 ml, Sterilized and refrigerated, Calcium Ionophore A23187 (Sigma Aldrich 52665-69-7) DAF-FM Di Acetate (Molecular Probes Life Technologies), and DAF-FM Di Aceatate was prepared as a stock solution (5 mM) in DMSO, divided into aliquots and stored at -20 °C, followed by dilution to the required concentration in HBSS buffer before use. EQUIPMENT: Neubauer chamber, Microtube centrifuges (1.5 mL), Micropipettors,10,100, and 1000 mL with corresponding tips, multimode reader (Tecan, Synergy-HT), inverted fluorescence microscope (Nikon, eclipse Ti-S), black flat bottom Microplates (96-well) (Corning 3603).
一般来说，4 氨基-5 甲基氨基-2'，7' - 二氟荧光素二乙酸酯（DAF-FM-DA）染料用于通过细胞成像检测生物系统中的一氧化氮。在本研究中，我们使用 96 孔板格式通过多模式读取器（或独立使用荧光分光光度计）定量使用 DAF-FM-DA 的一氧化氮，并可在荧光显微镜下观察到。否则，类似的研究将需要高端仪器。该方法已通过在过表达 NOS 同工型的 HEK 293T 细胞系中筛选 NOS 抑制剂进行了验证。我们观察到该方法非常简单易用，适应性强，灵敏度高，最重要的是它节省时间。试剂/工具：乙醇（70%[v/v]在蒸馏水中），Nω-硝基-L-精氨酸（l-NAME），7-硝基吲唑（7-NI）（Sigma，圣路易斯，密苏里州），HEK 293T 细胞系（国家细胞科学中心（NCCS），浦那，印度），DMEM（Himedia 实验室 Pvt），胎牛血清（FBS）（Invitrogen，卡尔斯巴德，加利福尼亚州），100U/mL 青霉素和 0.1mg/mL 链霉素在 5%CO2 气氛中。用以下成分制备 pH7.4 无酚红的 Hank's 平衡盐溶液：NaCl，8.0g，KCl，0.4g，CaCl，0.14g，MgSO·7HO，0.1g，MgCl·6HO，0.1g，NaHPO·2HO，0.06g，KHPO，0.06g，葡萄糖，1.0g，NaHCO，0.35g，HO，至 1000ml，消毒并冷藏，钙离子载体 A23187（Sigma Aldrich 52665-69-7）DAF-FM 二乙酸酯（Molecular Probes Life Technologies），并将 DAF-FM 二乙酰胺酯作为储备溶液（5mM）在 DMSO 中制备，分成等分试样，并储存在-20°C 下，然后在使用前在 HBSS 缓冲液中稀释至所需浓度。设备：纽巴氏室，微量离心机（1.5mL），微量移液器，10、100 和 1000mL 及相应的吸头，多模式读取器（Tecan，Synergy-HT），倒置荧光显微镜（尼康，eclipse Ti-S），黑色平底微孔板（96 孔）（康宁 3603）。
