
Description
There are five fluorescent control liposome products (Fluoroliposome®) for Clodrosome® (clodronate liposomes). All five fluorescent liposomes incorporate a lipophilic dye inside their membranes. They are insoluble in water; however, their fluorescence is easily detected when incorporated into membranes. DiI, DiO, DiD, DiR and DiA cover a wide range of excitation and emission wavelengths from 300s to 900s. DiI and DiO have fluorescence excitation and emission maxima separated by about 65 nm, facilitating two-color labeling. The emission spectrum of DiA is very broad, allowing it to be detected as green, orange or even red fluorescence depending on the optical filter used. DiI, DiO, DiD and DiR belong to the dialkylcarbocyanines family of compounds. The spectral properties of the dialkylcarbocyanines are largely independent of the lengths of the alkyl chains. Instead, they are determined by the heteroatoms in the terminal ring systems and the length of the connecting bridge. They have extremely high extinction coefficients, moderate fluorescence quantum yields and short excited state lifetimes in lipid environments (~1 ns). The fluorescence spectrum of each dye is shown below.
You can choose the Fluoroliposome® based on the type of the fluorescent equipment and filters that you use in your lab. Clodronate liposomes cannot be made fluorescent simply due to the potential for inaccurate and/or uninterpretable data being generated by labelled Clodrosome®. For more information, please refer to the technical note section.


Technical Information
Three-Color Fluoroliposome® Kit
Lipid Composition | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
---|---|---|---|
Total | 23 mg/ml | 35.1 mM | 100 |
L-alpha-Phosphatidylcholine | 18.8 | 24.3 | 70 |
Cholesterol | 4.2 | 10.9 | 30 |
Fluorescent Dye | Excitation/Emission (nm) | Concentration (mg/ml) | Concentration (mM) |
---|---|---|---|
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD)![]() | 644/665 | 0.0625 | 0.065 |
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiI)![]() | 549/565 | 0.0625 | 0.065 |
3,3'-Dilinoleyloxacarbocyanine Perchlorate (DiO)![]() | 484/501 | 0.0625 | 0.071 |
Buffer and Liposome Size | Specification |
---|---|
Buffer | Phosphate Buffered Saline |
pH | 7.4 |
Liposome Size | 1.5-2 µm |
Technical Notes
- The issue with fluorescent Clodrosome® has to do with the potential for inaccurate and/or uninterpretable data being generated by labelled Clodrosome®. When Clodrosome® induces macrophage apoptosis, the fluorescent lipid incorporated into the Clodrosome® is disrupted and metabolized in the phagolysosome will be dispersed among the residual apoptotic bodies which are subsequently phagocytosed by other macrophages. Therefore, fluorescent lipid may be detected in phagocytic cells which never phagocytosed Clodrosome® especially when FACS or fluoroscopy are utilized to detect fluorescent cells (FACS) or fluorescence levels in a tissue homogenate (fluoroscopy). Another potential artifact arises from fluorescent lipid remaining in the extracellular “garbage”, which has not yet been cleared by other phagocytes, generating a high background fluorescence. However, experienced confocal microscopist may be able to differentiate between the punctate fluorescence, resulting from fluorescent intact liposomes versus the more diffuse fluorescence characteristic of disrupted liposomes and some have successfully used fluorescent clodronate liposomes to visualize the cellular location of these liposomes by confocal microscopy in vivo [2]. A further complicating factor is that published data varies widely as to exactly when clodronate liposomes begin to induce apoptosis in macrophages. Mönkönnnen et al. show that macrophage death is measurable within the first hour after clodronate liposome treatment on RAW264 cells in vitro [3], while many others have reported no signs of macrophage apoptosis until several hours after treatment in vivo. The variability in the data is likely due to different liposomal formulations of clodronate as well as the vastly different experimental conditions. Therefore, as with most biological studies, especially those involving liposomes, the amount of time between treating the animal or cells with clodronate liposomes and the onset of apoptosis will need to be established in each experimental model. If the nature of the research demands that Clodrosome® be tracked rather than the control, Encapsula can provide DiI-labelled Clodrosome® upon request, and assuming that the Clodrosome® distribution can definitively be assessed prior to the onset of apoptosis, clear and valid data on the biodistribution of fluorescent Clodrosome® should be obtainable. Still, for most purposes, Fluoroliposome® (fluorescent control liposomes) will provide the required data with far fewer potential artifacts.
- When monitoring monocyte uptake in vivo in normal animals, the circulating monocytes may “disappear” or show reduced counts within the first 2 h post-injection due to margination of the monocytes post-liposome phagocytosis. These cells will re-enter the circulation within a few hours. Sunderkötter et al. demonstrate this phenomenon and discuss the behavior in detail. Also consider that circulating monocytes have a lifetime of about 24 h so labeled monocytes will be continually leaving the circulation, even in normal animals, due to aging of the monocytes [4].
- Liposomes may settle when left undisturbed for more than a few hours. Immediately prior to use, in order to ensure a homogeneous liposome suspension, slowly invert the vial several times until the suspension appears homogeneous by visual inspection. Vigorous or erratic shaking will not damage the liposomes but may induce foaming and bubble formation making it more difficult to accurately measure the desired dosage.
- If the personnel performing intravenous injections are not experienced in or familiar with, precautions for injecting larger volumes (~10% animal weight in ml), viscous liquids or particulate suspensions, consider having extra animals available in case serious injection-related adverse events occur. Dose control animals first to become familiar with large volume injections.
- When dosing intravenously, use standard precautions for dosing larger volumes to animals including the following: a) warm product to room temperature prior to dosing; b) ensure that all air bubbles are removed from the syringe prior to dosing. Intravenous injection of air bubbles may result in air emboli which can kill or seriously injure animals; c) inject product at a slow, steady rate of no more than 1 ml/min; d) decrease infusion rate if animals display any atypical reactions such as unusual agitation.
- Infusion-related adverse reactions usually involve the animal gasping for air or other seizure-like movements. Animals often recover with no apparent permanent injury, but any potential effects on experimental results must be assessed by the researcher.
- Liposomes should be kept at 4°C and NEVER be frozen.
Dosage
Appearance
Fluoroliposome®-DiD, Fluoroliposome®-DiI, and Fluoroliposome®-DiO are blue, pink and yellow liquid suspensions, respectively. These Fluoroliposome® products are made of large micron size multilamellar liposomes. Due to their large size, some liposomes might settle to the bottom of the vial. If left sitting idle in the refrigerator, they will phase separate and form pellets in the bottom of the vial, leaving a clear solution on top. Therefore, the vial should be shaken to form a homogeneous solution prior to use.
Educational Videos
Ordering/Shipping Information
- All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
- Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
- Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
- We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
- Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
- If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
- Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.
Storage and Shelf Life
Storage
Fluoroliposome® products should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. ENS is not responsible for results generated by frozen product.
Shelf Life
Fluoroliposome® products are made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 60 days of the manufacturing date.
References and background reading
1. Polfliet MM, Goede PH, van Kesteren-Hendrikx EM, van Rooijen N, Dijkstra CD, van den Berg TK. A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system. J. Neuroimmunol. 2001 Jun 1;116(2):188–95.
2. Mönkkönen J, Liukkonen J, Taskinen M, Heath TD, Urtti A. Studies on liposome formulations for intra-articular delivery of clodronate. Journal of Controlled Release. 1995 Aug;35(2–3):145–54.
3. Sunderkötter C, Nikolic T, Dillon MJ, van Rooijen N, Stehling M, Drevets DA, Leenen P. Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response. J Immunol. 2004 Apr 1;172(7):4410–7.
4. Nagai H, Kuwahira I, Schwenke DO, Tsuchimochi H, Nara A, Ogura S, Sonobe T, Inagaki T, Fujii Y, Yamaguchi R, Wingenfeld L. Pulmonary macrophages attenuate hypoxic pulmonary vasoconstriction via β3AR/iNOS pathway in rats exposed to chronic intermittent hypoxia. PLoS One. 2015 Jul 1;10(7):e0131923.
5. Zhu Y, Soderblom C, Krishnan V, Ashbaugh J, Bethea JR, Lee JK. Hematogenous macrophage depletion reduces the fibrotic scar and increases axonal growth after spinal cord injury. Neurobiology of disease. 2015 Feb 28;74:114-25.
6. Yun MH, Davaapil H, Brockes JP. Recurrent turnover of senescent cells during regeneration of a complex structure. Elife. 2015;4:e05505.
7. Arwert EN, Harney AS, Entenberg D, Wang Y, Sahai E, Pollard JW, Condeelis JS. A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation. Cell reports. 2018 May 1;23(5):1239-48.
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其检测原理为:在正常的活细胞中,磷脂酰丝氨酸(phosphotidylserine,PS)位于细胞膜的内侧,但在早期凋亡的细胞中,PS 从细胞膜的内侧翻转到细胞膜的表面,暴露在细胞外环境中。Annexin-Ⅴ(膜联蛋白-V)是一种分子量为35-36KD的Ca2+ 依赖性磷脂结合蛋白,能与PS高亲和力结合。可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。
操作步骤:
1.从室温平衡20min后的铝箔袋中取出所需板条,剩余板条用自封袋密封放回4℃。
2.设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL;
3.样本孔中加入待测样本50μL;空白孔不加。
4.除空白孔外,标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL,用封板膜封住反应孔,37℃水浴锅或恒温箱温育60min。
5.弃去液体,吸水纸上拍干,每孔加满洗涤液(350μL),静置1min,甩去洗涤液,吸水纸上拍干,如此重复洗板5次(也可用洗板机洗板)。
6.每孔加入底物A、B各50μL,37℃避光孵育15min。
7.每孔加入终止液50μL,15min内,在450nm波长处测定各孔的OD值。
不知道发在这里合适不,实在是求助无门啊!版主手下留情。
想请问下那个公司有专门的蛋白质荧光标记试剂盒出售。最好的是CY5的,我准备做三标。性价比越高越好
那些做过的前辈们指导一下。
2、 血浆:EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。
3、 细胞上清液:1000×g离心10分钟去除颗粒和聚合物。
4、 组织匀浆:将组织加入适量生理盐水捣碎。1000×g离心10分钟,取上清液。
5、 保存:如果样品不立即使用,应将其分成小部分-70℃保存,避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒,检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。
此IBL试剂盒能用于小鼠血清,EDTA血浆,细胞上清中白介素-6的定量检测 试剂盒成分 1 预包被板: 抗小鼠白介素-6兔子IgG,亲合纯化 96T 2 酶标记抗体: (30倍浓缩)HRP标记抗小鼠白介素-6兔子IgG,亲合纯化 0.4mL x 1 3 标准品: 重组小鼠白介素-6 0.5mL x 2 4 EIA缓冲液: 含1% BSA, 0.05%吐温20 BPS 30mL x 1 5 标记抗体稀释液: 含1% BSA, 0.05%吐温20 BPS 12mL x 1 6 显色剂: TMB底物液 15mL x 1 7 终止液: 1N硫酸 12mL x 1 8 浓缩洗涤液: (40倍浓缩) 含1% BSA, 0.05%吐温20 BPS 50mL x 1 操作说明 1实验所需器材(但试剂盒没有提供) 酶标仪(450nm) 微移液管及其吸嘴 量筒及烧杯 去离子水 冰箱(4°C) 坐标纸(log/log) 吸水纸 试管(用于标准品稀释) 温育箱(37°C ± 1°C) 洗瓶 (用于洗板) 一次性试剂管(用于浓缩酶标记抗体和显色剂)向左转|向右转