Fluorescent-DiA Macrophage Depletion Kit
Macrophage depletion kits are composed of two vials; one vial of Clodrosome® (Clodronate liposomes) and one vial of Encapsome® (control liposomes containing no drug). The volume of the macrophage depletion kit represents the volume of each reagent individually. For example, the 5-ml macrophage depletion kit means 5 ml of Clodrosome® and 5 ml of Encapsome®. Each reagent in the kit can also be purchased individually.
Clodrosome® is a multilamellar liposome suspension in which clodronate is encapsulated in the aqueous compartments of the liposomes. Encapsome® is formulated and prepared identically to Clodrosome® except that clodronate is not added to the liposomes. The liposomes are filtered through 2 μm polycarbonate membranes to ensure that the larger particles, which may be toxic to animals, are removed from the suspension. Both are prepared and packaged under sterile conditions. When animals or cells are treated with Clodrosome®, phagocytic cells recognize the liposomes as invading foreign particles and proceed to remove the liposomes from the local tissue or serum via phagocytosis. The liposomes then release clodronate into the cytosol, resulting in cell death. Non-encapsulated clodronate cannot cross the cell membrane to initiate cell death.
Control liposomes (Encapsome®) are recognized and phagocytosed by the same mechanism as Clodrosome®. Since the control liposomes do not contain clodronate, the phagocytic cells are not killed. However, phagocytes do respond to the ingestion of control liposomes by cytokine secretion, temporary suspension of phagocytic activity and other responses described in the literature.
m-Clodrosome® and m-Encapsome® are mannosylated reagents that are specifically formulated to efficiently target the macrophages in central nervous systems and macrophages that contain more mannose receptors. For more information about these reagent see here.
Fluorescent liposomes (Fluoroliposome®) suitable for macrophage targeting and tracking are available. They can contain five different fluorescent dyes (DiA, DiD, DiI, DiO and DiR), which covers the entire spectrum. Fluorescent liposomes come in standard and mannosylated form. For more information see here.
Clodrosome® Liposomal Clodronate Suspension
|Lipid Composition||Concentration (mg/ml)||Concentration (mM)||Molar Ratio Percentage|
|Total||23 mg/ml||35.1 mM||100|
|Clodronate ((Dichloro-phosphono-methyl)phosphonate), Disodium Salt||18.4* mM|
|* Depending on the type of the clodronate salt, its concentration (mg/ml) varies. If tetra hydrate salt is used, the concentration of the encapsulated drug will be ~7 mg/ml, and if a non-hydrated salt is used, the concentration will be ~5 mg/ml.|
|Lipid Composition||Concentration (mg/ml)||Concentration (mM)||Molar Ratio Percentage|
|Total||23 mg/ml||35.1 mM||100|
|Fluorescent Dye||Excitation/Emission (nm)||Concentration (mg/ml)||Concentration (mM)|
|4-(4-(Dihexadecylamino)styryl)-N-methylpyridinium Iodide (DiA)||456/590||0.0625||0.0794|
|Buffer and Liposome Size||Specification|
|Buffer||Phosphate Buffered Saline|
|Liposome Size||1.5-2 µm|
- 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® that 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 . 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 , 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 .
- When animals or cells are treated with Clodrosome®, phagocytic cells recognize the liposomes as invading foreign particles and proceed to remove the liposomes from the local tissue or serum via phagocytosis. The liposomes then release clodronate into the cytosol resulting in cell death. Unencapsulated clodronate cannot cross the cell membrane to initiate cell death.
- Encapsome® control liposomes are recognized and phagocytosed by the same mechanism as Clodrosome®. Since the control liposomes do not contain clodronate, the phagocytic cells are not killed. However, phagocytes do respond to the ingestion of the control liposomes by cytokine secretion, temporary suspension of phagocytic activity and other responses described in the literature.
- The product must be removed from the vial using sterile technique. Do not use if sterility is compromised. This is particularly important if a single vial is accessed multiple times over several weeks. The product should not be used more than 60 days after receipt, even if unopened.
- 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.
- Within hours after systemic administration of Clodrosome®, animals begin to lose important components of their immune system. Standard animal handling and housing protocols are not suitable for immunocompromised animals. Even when such precautions are taken, monitor the general health of each animal for opportunistic infections unrelated to the experimental protocol. There is no inherent toxicity to the product at the recommended dose levels.
- 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.
Clodrosome® is a white milky suspension, and Fluoroliposome®-DiA is a yellow liquid suspension, both made of large micro 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, Fluoroliposome®-DiA will phase separate and form pellets in the bottom of the vial, leaving a clear solution on top. Clodrosome® might do the same only not as severely. Therefore, both should be gently shaken not to form bubbles but to form a homogeneous solution prior to use.
- 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
Clodrosome® and Fluoroliposome® 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. If the suspension is frozen, clodronate can be released from the liposomes thus limiting its effectiveness in depleting macrophages. ENS is not responsible for results generated by frozen product.
Clodrosome® and Fluoroliposome® 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.
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.
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9. Zhu Z, Ding J, Ma Z, Iwashina T, Tredget EE. Systemic depletion of macrophages in the subacute phase of wound healing reduces hypertrophic scar formation. Wound Repair and Regeneration. 2016 Jul 1;24(4):644-56.
10. Haque MR, Lee DY, Ahn CH, Jeong JH, Byun Y. Local co-delivery of pancreatic islets and liposomal clodronate using injectable hydrogel to prevent acute immune reactions in a type 1 diabetes. Pharmaceutical research. 2014 Sep 1;31(9):2453-62.
11. Mayo L, Cunha AP, Madi A, Beynon V, Yang Z, Alvarez JI, Prat A, Sobel RA, Kobzik L, Lassmann H, Quintana FJ. IL-10-dependent Tr1 cells attenuate astrocyte activation and ameliorate chronic central nervous system inflammation. Brain. 2016 May 31;139(7):1939-57.
12. Kermanizadeh A, Chauché C, Balharry D, Brown DM, Kanase N, Boczkowski J, Lanone S, Stone V. The role of Kupffer cells in the hepatic response to silver nanoparticles. Nanotoxicology. 2014 Aug 31;8(sup1):149-54.
13. Nandi B, Shapiro M, Samur MK, Pai C, Frank NY, Yoon C, Prabhala RH, Munshi NC, Gold JS. Stromal CCR6 drives tumor growth in a murine transplantable colon cancer through recruitment of tumor-promoting macrophages. Oncoimmunology. 2016 Aug 2;5(8):e1189052.