I did not observe macrophage depletion, what can be the reason?

Failure of activity of clodronate liposomes can be due to various reasons:

1. The expiry date has passed: activity cannot be ensured after the expiry date has passed;

2. Storage at extreme temperatures: clodronate liposomes should be stored between 4 and 8 degrees Celsius. Freezing of liposomes or temperatures above 30 degrees Celsius can affect the membrane’s stability and reduce clodronate liposome activity;

3. Dilution of the liposomal suspension: this affects the osmotic pressure in the suspension, which may cause instability of the liposomal membrane;

4. The administration protocol is inadequate: protocols should be chosen carefully and depend on multiple factors, for instance:

1. the type of macrophages you intend to deplete (e.g. Kupffer cells, microglia, alveolar macrophages, osteoclasts). For example, alveolar macrophages can be targeted by intratracheal administration, whilst the interstitial macrophages on the other side of the endothelial/epithelial barrier will not be targeted by this administration route;
2. whether you intend to deplete them short- or long-term;
3. the weight of the animal: larger animals require more clodronate liposomes in order to establish depletion.

5. The liposomal suspension was not resuspended before use: liposomes tend to precipitate during storage, make sure to resuspend by gently shaking before usage to prevent injecting only the PBS solution without the liposomes;

6. Something went wrong whilst injecting: it is important to verify whether the injection is successful. For instance, if you see a bulb forming during intravenous injection in the tail vein, it might be the case that you missed the tail vein. When injecting intraperitoneally, make sure that you inject the liposomal suspension into the intraperitoneal cavity (and not in the intestine). To verify if the liposomes have reached your desired location, fluorescent DiI liposomes can be used;

7. Inadequate ways of measuring depletion: the right markers need to be chosen to establish whether depletion has been successful. In addition, you need to take into account that depletion and repopulation kinetics of macrophages differ per administration protocol and (animal) model.