FITC-Dextran (MW 2000000)   中文名称: FITC-dextran

Fluorescent Dye

异硫氰酸荧光素 (FITC) 葡聚糖荧光染料（Ex=495 nm；Em=525 nm）被称为 FITC-葡聚糖（MW 500000）。为了研究细胞封闭的早期和晚期阶段并识别热休克引起的细胞损伤，可以使用 FITC-葡聚糖 (MW 500000) 作为标记物。对于细胞通透性研究，例如血脑屏障通透性和血脑屏障破坏程度的评估，使用 FITC-葡聚糖 (MW 500000)。储存：避免阳光直射。

标准操作流程
（以下为我们建议的实验方案，请根据具体应用需求进行调整。）

肠道屏障功能检测 [5]

1. 禁食处理
实验前使小鼠禁食4小时。
2. FITC-葡聚糖（分子量70,000）给药
采用灌胃方式给予FITC-葡聚糖（0.6 mg/g体重）。
3. 荧光强度检测
给药后4小时内测定血清荧光强度。

检测参数：
激发波长(Ex)：490 nm
发射波长(Em)：520 nm

关键说明：
本实验通过检测FITC-葡聚糖向循环系统的渗漏来评估肠道通透性。
荧光强度越高表明肠道屏障完整性受损越严重。
需确保规范的禁食处理以避免食物消化对实验结果的干扰。

（注：实验条件可根据具体研究需求进行相应调整。）

细胞培养与FITC-葡聚糖负载（参见注释2）[3]
1. 将培养于细胞培养基中的人源成纤维细胞置于37°C、5% CO2的湿润培养箱中培养，每周传代一次（参见注释3）。
2. 用胰酶消化细胞，计数后以9000个细胞/cm²的密度接种于直径35mm细胞培养皿中（参见注释4）。建议每组样品设置至少三个复孔，并建立包含五个pH值（如pH 4.05、4.5、5.0、5.5和6.0）的标准曲线（参见注释5）。同时设置未染色样本对照。
3. 配制1 mg/ml的FITC-葡聚糖细胞培养基溶液，使用0.22μm孔径的亲水性聚醚砜膜针头过滤器进行除菌过滤。
4. 制备终浓度为0.1 mg/ml的FITC-葡聚糖细胞培养基（参见注释6）。
5. 吸除原培养液，加入1ml含FITC-葡聚糖的新鲜培养基，置于37°C、5% CO2培养箱中孵育3天（参见注释7）。

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溶酶体pH值检测准备（参见注释7）[3]
1. 通过吸除培养基并更换新鲜培养液，使FITC-葡聚糖定位于溶酶体，继续培养2小时（参见注释8）。如需验证，可用荧光显微镜观察FITC-葡聚糖是否呈溶酶体特征性点状分布（图2）。此期间可同时加入溶酶体膜通透性诱导剂或抑制剂（参见注释9）。
2. 孵育结束后，用胰酶消化细胞并转移至离心管。
3. 300×g离心5分钟，吸除上清。
4. 用1ml室温PBS洗涤细胞，再次300×g离心5分钟。
5. 弃PBS，将样本置于冰上。
6. 按每个标准样品0.5ml的体积配制不同pH值的Britton-Robinson缓冲液。添加终浓度50mM的叠氮化钠和2-脱氧葡萄糖，以及终浓度10μM的尼日利亚菌素（参见注释10）。缓冲液需保持冰浴状态。

[1]. Fluorescein isothiocyanate-dextran can track apoptosis and necrosis induced by heat shock of peripheral blood mononuclear cells and HeLa cells. Open Biological Sciences Journal, 2015, 1(1).

[2]. Fluorescein Isothiocyanate (FITC)-Dextran Extravasation as a Measure of Blood-Brain Barrier Permeability. Curr Protoc Neurosci. 2017 Apr 10;79:9.58.1-9.58.15.

[3]. Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran (MW 2000000) Loaded Cells. Methods Mol Biol. 2017;1594:179-189.

[4]. Cdc42 activates paracellular transport in polarised submandibular gland cells. Arch Oral Biol. 2021 Dec;132:105276.

[5]. ACE2 contributes to the maintenance of mouse epithelial barrier function. Biochem Biophys Res Commun. 2020 Dec 17;533(4):1276-1282.

Critical Parameters [2]
\n1.\tIt is important to use a correct dose of ketamine/xylazine cocktail for anesthesia. The dose should be sufficient for the rat to remain at a surgical plane of anesthesia without being lethal. For good perfusion to occur the heart should be beating at regular intervals when the heparin is injected into the left ventricle. The heparin injection is given in order to prevent platelet aggregation within the blood vessels and to provide a free flow of blood without any back pressure during perfusion, thereby minimizing rupture of small blood vessels and capillaries.
\n2.\tThe flow of the perfusion solution needs to be maintained at a rate that is not too high as i
t would increase pressure on small capillaries causing them to rupture. On the other hand pressure has to be maintained sufficiently to perfuse the smallest diameter capillaries.\n3.\tThe incision made in the right atrium for blood outflow has to be sufficiently large (~0.5 cm) for the blood to empty freely without building back pressure.
\n4.\tThe concentration of FITC-Dextran (MW 2000000) solution used in perfusion should be high enough to overcome the dilution by blood in the vessels during perfusion. The optimal concentration of FITC-Dextran (MW 2000000) to use in perfusion should be empirically determined by the experimenter based on the weight of the animal.
\n5.\tIt is important to ensure that FITC-Dextran (MW 2000000) powder is completely solubilized prior to perfusion and is cold.
\n6.\tRats must be decapitated and the brain removed immediately after perfusion with FITC-Dextran (MW 2000000) is completed. This would minimize artificial leakage of FITC-Dextran (MW 2000000) from the vessels in the absence of active perfusion by the pump.\n7.\tCryoprotection of the brain is essential for preventing sudden osmotic changes in cells during flash freezing that would burst the cell wall, damage tissue, and contribute to artificial leakage of FITC-Dextran (MW 2000000).
\n8.\tDo not immerse brains in 2-methylbutane for longer than 5 min as it causes fracturing of the tissue.
\n9.\tUsing subbed slides for mounting tissue sections helps the tissue adhere to and flatten on the glass slide. Additionally, it prevents the sections from lifting off the slide when incubating in DRAQ5.
\n10.\tIt is essential to equilibrate the brains to the temperature of the cryostat for at least 2 hr. Frequently, the outside of the brain is at a different temperature compared to the inner regions and this interferes with obtaining consistent slice thickness and also affects the quality of the section.
\n11.\tThe temperature at which the sections are sliced is important. Sectioning when it is too warm will make the tissue clump and stick to itself. In contrast, if the cryostat temperature is too cold, the tissue will crack and roll up tightly and make it difficult to unroll without causing damage to the tissue. Appropriate temperature setting of the cryostat depends on ambient temperature and humidity, and should be determined prior to sectioning of the area of interest by assessing the quality of sections from a region of the brain that is not of interest.
\n12.\tAfter sectioning, the tissue slices should be carefully and gently transferred from the cryostat blade using a thin brush and placed on the slide, as the blood vessels can break due to mechanical agitation and FITC can leak out of the capillaries.
\n13.\tLow-light conditions should be maintained when working with the FITC-Dextran (MW 2000000) perfused brain tissue to minimize the degradation of fluorescence due to quenching by ambient bright light. Low ambient light and covering the brains and tissue with foil help maintain fluorescence.
\n14.\tSlide mounted tissue sections need to be completely rehydrated in PBS (with or without DRAQ5) prior to Fluoromount-G application and coverslipping. Fluoromount-G is a mounting media that works well on wet tissue but its application to dry dehydrated sections forms numerous small air bubbles during setting of the mounting media.
\n15.\tDo not wash the section after incubation with DRAQ5 as it could wash out FITC-Dextran (MW 2000000) and decrease the DRAQ5 signal making it difficult to identify the correct focal plane during imaging.
\n16.\tBoth control and treated groups need to be imaged in every session. This would take into account any day-to-day variability or unintended changes in the confocal microscope and treatment of the slides.
\n17.\tSame brain areas/regions should be imaged in control and experimental groups in one imaging session rather than imaging all the brain regions of one group followed by a different group.
\n18.\tOnce the imaging parameters are established, the investigator performing the analyses should be blind to the treatment conditions in order to eliminate introduction of bias to imaging measures.\n\n

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\nTroubleshooting
\n1.\tToo dark or no visible capillaries in a control rat can be due to poor perfusion
\na.\tLiver clearing can be used as an indicator of perfusion. The liver should begin to clear out in 5 – 10 s after starting perfusion. If this does not happen, readjust the position of the 16 G needle in the ventricle making sure that the beveled tip is not against the wall of the heart or septum.
\nb.\tIncrease the volume of FITC-Dextran (MW 2000000) perfused. The amount of FITC-Dextran (MW 2000000) suggested (12 mL) is for rats weighing between 250–300 g. Heavier rats may require a greater volume of perfused FITC-Dextran (MW 2000000) to fill the small capillaries in the brain.
\nc.\tAn increase in the pump flow rate could also be tried, however there is a concern that higher speeds and therefore, higher pressure, could cause brain capillaries to rupture and leak FITC-Dextran (MW 2000000), leading to false positives for BBB disruption. Keep the pump rate consistent for all control and experimental groups so that changes in the rate of the pump do not unintentionally cause variability in BBB disruption.

\n2.\tBright fluorescent speckles all over the section and slides during imaging
\na.\tToo much disruption of the tissue section when slicing or placing onto subbed slides can rupture blood vessels and capillaries causing FITC-Dextran (MW 2000000) to leak out.
\nb.\tKimwipes or other lab tissues can leave behind specks and debris that fluoresce at different wavelengths of light and should not be used for cleaning slides. A soft microfiber cloth is a better alternative for wiping slides clean.
\nc.\tThe freezing process could also lead to bright fluorescent specks over the tissue. Incubation in the 2-methylbutane for longer than 5 min could cause tissue dehydration and rupture of cell membranes.\n\n

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\n\nThe blood-brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption. A detailed protocol is presented for assessing the integrity of the BBB by transcardial perfusion of a 10,000 Da FITC-labeled dextran molecule and its visualization to determine the degree of extravasation from brain microvessels. [2]

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\n\nThe acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization. [3]\n

C21H13NO6S.XUNSPECIFIED

2000000

60842-46-8

Yellow to orange solid powder

C1=C(O)C=CC2C3(OC(=O)C4=CC=CC=C34)C3=C(C=C(ONC(=O)S)C=C3)OC1=2

FITC-Dextran (MW 2000000)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

H2O : ~50 mg/mL

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。