Fluorescent Dye

1.工作溶液的配制
1.1储备溶液的制备。为了获得1mM储备溶液，将1mg 2-NBDG溶解在2.92mL DDH2O中
1.2工作溶液的制备：用预热的无血清细胞培养基或PBS稀释储备溶液，制备10-200μM的2-NBDG工作溶液
注：请根据您的具体需要调整2-NBDG工作溶液的浓度
2.细胞染色
2.1悬浮细胞：离心收集细胞，加入PBS洗涤两次，每次5分钟
贴壁细胞：弃掉培养基，加入胰蛋白酶消化细胞。离心后，丢弃上清液并用PBS洗涤两次，每次5分钟
2.2加入1mL 2-NBDG工作溶液，在室温下孵育5-60分钟
2.3 400 g，4℃离心3-4分钟，弃去上清液
2.4 加入PBS洗涤细胞两次，每次5分钟
2.5 用1mL无血清培养基或PBS重悬细胞后，在显微镜下观察。如果进行了活力测试，则使用ELISA酶联免疫吸附测定（ELISA）读取器记录540/570 nm处的光密度（O.D.）。使用对照比例计算细胞活力，并相对于药物的对数浓度绘制以计算IC50。

通过荧光成像，在携带人类癌症异种移植物肿瘤的小鼠中检测到荧光2-NBDG摄取增加的癌症循环细胞，这表明2-NBDG作为超代谢循环癌症细胞荧光成像的示踪剂在临床上的应用[3]。

循环乳腺癌症细胞对荧光2-NBDG吸收增加的荧光成像从肿瘤细胞植入后1周开始，通过穿刺小鼠隐静脉收集小鼠血液样本（100μL/小鼠）。将含有循环乳腺癌症细胞的血液样品与购自Invitrogen的荧光葡萄糖类似物2-NBDG在37°C的黑暗培养箱中以5μg/100μL血液的剂量孵育30分钟。随后，在来自供应商的方案中，用与抗上皮细胞粘附分子（EpCAM）IgG缀合的磁珠收获循环乳腺癌症细胞，随后用细胞摄取荧光2-NBDG对循环乳腺癌症细胞进行荧光成像。简言之，在与2-NBDG孵育结束时，将1μL 1%的磁珠悬浮液添加到100μL的血液样品中，然后在4°C下孵育30分钟，轻轻摇晃，以促进磁珠与循环的乳腺癌症细胞结合。随后，通过磁分离架磁分离从血液中收集循环的乳腺癌症细胞，用PBS洗涤3次，并在100μl PBS中再次悬浮循环的乳腺癌症细胞后转移到96细胞板中。在配备488nm滤光片的荧光显微镜（Olympus）下检查循环的乳腺癌症细胞对2-NBDG的摄取。具有来源于荧光2-NBDG的细胞摄取的荧光信号的大细胞被计数为高代谢循环乳腺癌症细胞，相比之下，小尺寸的正常小鼠血细胞（淋巴细胞和RBC）没有或很少显示2-NBDG荧光信号。通过用手动细胞计数器对96孔板的孔的整个区域进行视觉扫描，获得血液样品中高代谢循环乳腺癌症细胞的总数。实验重复了三次[3]。

2-NBDG摄取测定的染色实施例1：2-NBDG可用作监测活细胞中葡萄糖摄取的荧光指示剂
1.将细胞与2-NBDG（50μM；30分钟；37℃；5%CO2）在无葡萄糖DMEM中孵育
2.彻底清洗细胞并通过显微镜测量荧光

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2-NBDG摄取测定的染色实施例2：2-NBDG可用作监测活细胞中葡萄糖摄取的荧光指示剂
1.用含有2-NBDG的低血糖DMEM（150μg/mL；60分钟；37℃）孵育细胞
2.彻底清洗细胞并通过显微镜测量荧光

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2-NBDG摄取测定的染色实施例3：2-NBDG可用作监测活细胞中葡萄糖摄取的荧光指示剂
1.将细胞在低葡萄糖DMEM中与2-NBDG（150μg/mL；60分钟；37℃）孵育
2.彻底清洗细胞并通过显微镜测量荧光

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2-NBDG摄取测定的染色实施例4：2-NBDG可用作监测活细胞中葡萄糖摄取的荧光指示剂
1.用2-NBDG（100μM；30分钟）孵育细胞
2.彻底清洗细胞并通过显微镜测量荧光

Procedures:[4]
Human breast cancer cells were implanted in the mammary gland fat pad of athymic mice to establish orthotopic human breast cancer xenografts as a mouse model of circulating breast cancer cells. Near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DeoxyGlucosone 750 (2-DG 750) was conducted to assess glucose metabolism of xenograft tumors. Following incubation with fluorescent 2-NBDG, circulating breast cancer cells in the blood samples collected from the tumor-bearing mice were collected by magnetic separation, followed by fluorescence imaging for 2-NBDG uptake by circulating breast cancer cells, and correlation of the number of hypermetabolic circulating breast cancer cells with tumor size at the time when the blood samples were collected.

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Results: [4]
Human breast cancer xenograft tumors derived from MDA-MB-231, BT474, or SKBR-3 cells were visualized on near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DG 750. Hypermetabolic circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in the blood samples from tumor-bearing mice and visualized by fluorescence imaging, but not in the blood samples from normal control mice. The number of hypermetabolic circulating breast cancer cells increased along with growth of xenograft tumors, with the number of hypermetabolic circulating breast cancer cells detected in the mice bearing MDA-MB231 xenografts larger than those in the mice bearing BT474 or SKBR-3 xenograft tumors.

[1]. Yamada K, et al. A real-time method of imaging glucose uptake in single, living mammalian cells. Nat Protoc. 2007;2(3):753-62.
[2]. Zou C, et al. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods. 2005 Sep 30;64(3):207-15.
[3]. J Fluoresc. 2013 Jan;23(1):213-20. doi: 10.1007/s10895-012-1136-z.

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.[1]

C12H14N4O8

342.26200

342.081

C, 42.11; H, 4.12; N, 16.37; O, 37.40

186689-07-6

2376921-70-7 (1-NBDG)

6711157

Typically exists as light yellow to orange solids at room temperature

1.750±0.06 g/cm3

707.6±70.0 °C at 760 mmHg

381.7±35.7 °C

0.0±2.4 mmHg at 25°C

1.770

-0.41

186.92

5

11

7

24

449

4

O=C[C@H](NC1=CC=C([N+]([O-])=O)C2=NON=C21)[C@H]([C@@H]([C@@H](CO)O)O)O

QUTFFEUUGHUPQC-ILWYWAAHSA-N

InChI=1S/C12H14N4O8/c17-3-6(11(20)12(21)8(19)4-18)13-5-1-2-7(16(22)23)10-9(5)14-24-15-10/h1-3,6,8,11-13,18-21H,4H2/t6-,8+,11+,12+/m0/s1

2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose

2-NBDG; 2NBDG; 2 NBDG

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~5 mg/mL (~14.61 mM)

配方 1 中的溶解度: 3.33 mg/mL (9.73 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。