| Storage | 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. |
| Appearance | Blue liquid suspension 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, 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. |
| Description | There are five fluorescent control liposome products for clodronate liposomes (CDECLD-1686). 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). |
| Lipid composition | L-α-Phosphatidylcholine (PC)/Cholesterol (70/30 molar ratio) |
| Liposome Size | 100 nm |
| Lipid Molar Concentration | 35.1 mM |
| Fluorophore | DiD |
| Fluorophore Concentration | 0.0625 mg/mL (0.065 mM) |
| Excitation | 644 nm |
| Emission | 665 nm |
| Buffer | Phosphate Buffered Saline, pH 7.4 |
| Shelf-life | 60 days |
| Technical Notes | The issue with fluorescent Clodronate Liposomes has to do with the potential for inaccurate and/or uninterpretable data being generated by labelled Clodronate Liposomes. When Clodronate Liposome (CDECLD-1686) induces macrophage apoptosis, the fluorescent lipid incorporated into the Clodronate Liposome (CDECLD-1686) 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 Clodronate Liposome (CDECLD-1686) 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 microscopists 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. 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 Clodronate Liposome (CDECLD-1686) be tracked rather than the control, We can provide DiI-labelled Clodronate Liposome upon request, and assuming that the Liposomal Clodronate distribution can definitively be assessed prior to the onset of apoptosis, clear and valid data on the biodistribution of fluorescent Clodronate Liposome should be obtainable. Still, for most purposes, these 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.
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; 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. |
| Lipid Mass Concentration | 23 mg/mL |
Datasheet