If you have the knowledge and facilities to prepare liposomes in your own laboratory, you can follow the detailed description of the technique in J.Immunol.Meth. 174 p.83-93,1994. The 'Roche' clodronate was used in a concentration of 2.5 grams per 10 ml. of aqueous solution (usually PBS). Please be aware that other industrial sources of clodronate are available and at moment we use clodronate from an industrial provider. Allthough different providers of clodronate give exactly the same specifications according to their product information sheets, there may be small differences in solubility. The industrial form that we used in our original studies (e.g. Van Rooijen, N.  and Van Nieuwmegen, R.  1984.  Elimination of  phagocytic  cells in the spleen after intravenous injection of liposome encapsulated  dichloromethylene-diphosphonate:  An  enzyme-histochemical  study.  Cell  Tissue Res. 238, 355-358)  could be used in a maximum concentration  of ca 1.9-2.0 grams per 10 ml of aqueous solution. If you do not know the origin of your clodronate, we recommend to try at first the lower concentration. At moment there is no explanation for this difference, but the resulting encapsulation efficacy of both forms of clodronate is not markedly different. The final suspension of the present clodronate liposomes does contain ca. 5 milligrams of clodronate per ml of the suspension (was 7 milligrams for the Roche clodronate). The clodronate solution remaining after the encapsulation procedure, can be reused by replenishing with fresh stock solution (only 1-2 % of the clodronate is encapsulated). To this purpose the final liposome suspension should not be diluted before the first centrifugation (washing) step. About 90% of the original clodronate solution can be recovered in this way. If as a consequence, liposomes are not spun down during the centrifuge step, they have to be collected from the surface. Keep the liposomes at a temperature 4 °C, and resuspend before administration to the animals.

For the most recent publications on the preparation of clodronate liposomes, see: 

Van Rooijen, N., Van Kesteren-Hendrikx, E., 2003. 'In vivo' depletion of macrophages by liposome-mediated 'suicide'.. Methods in Enzymology. vol. 373. pp. 3-16  &  Van Rooijen, N., Hendrikx, E. 2010. Liposomes for specific depletion of macrophages from organs and tissues. Methods Mol. Biol. 605; 189-203

Whether or not clodronate liposomes will be spun down (or precipitate) or will end up on top of the suspension depends on the specific gravity of the liposomes compared to the surrounding solution in which the suspension was made.
Please be aware that the specific gravities of the clodronate liposomes and their surrounding solution differ only marginally. The slightest difference can decide which of both will be on top and which of them will end up on the bottom.

Given that it is possible to limit the diameter of liposomes at a max. size, we are using Millipore filters at 3 microns in our standard preparations. Given also that we found (somewhere in the past) larger clodronate liposomes to be more efficacious than smaller ones, we did choose the max diameter that will not lead to problems in most laboratory animals used. 

Nevertheless we did use smaller liposomes in the only experiments in men up to now (a small clinical trial: Barrera, P., Blom, A., Van Lent, P.L.E.M., Van Bloois, L., Storm, G., Beijnen, J., Van Rooijen, N., Van De Putte, L.B.A., Van Den Berg, W.B. 2000. Synovial macrophage depletion with clodronate containing liposomes in rheumatoid arthritis. Arthritis. Rheum. 43; 1951-1959). This is because these filters did limit microbial contamination as much as possible. See for the problematic sterilization of liposomes FAQ (frequently asked questions).


Clodronate is currently purchased from an industrial provider

Materials for preparation of liposomes are:

Phosphatidylcholine (LIPOID E PC) is purchased from Lipoid GmbH, Ludwigshafen,Germany.

Cholesterol is purchased from SIGMA Chem.Co. USA.


       There is only one parameter that is important. The present suspension of liposome encapsulated clodronate is containing ca. 5 mg of clodronate per 1 ml of the suspension. The dimensions and other parameters of liposomes in the suspension are varying (not between different suspensions, but in one single suspension). Given that the clodronate enters the macrophage with the liposomes as Trojan horses, their dimensions are not important, only the total amount of encapsulated clodronate injected.

Useful references: (Full titles and authors can be found under references)

  • First Publication:
    For the first description of the approach, see: Van Rooijen, N. & Van Nieuwmegen, R. 1984; Cell Tissue Res.238; 355-358.
  • Preparation:
    For reviews on preparation of liposome-encapsulated clodronate, see e.g.: Van Rooijen, N. & Sanders, A. 1994; J.Immunol. Meth. 174; 83-93.  OR: Van Rooijen, N. & Van Kesteren-Hendrikx, E. 2003; Meth. Enzymol. 373; 3-16. OR: Van Rooijen, N. & Hendrikx, E. 2010. Methods Mol. Biol. 605; 189-203.
  • Applications:
    For reviews on applications of the approach, see: Van Rooijen, N., Bakker, J. & Sanders, A. 1997; Trends in Biotech. 15; 178-185, and Van Rooijen, N. & Van Kesteren-Hendrikx, E. 2002; J. Liposome Research 12; 81-94.
  • Alternatives for Clodronate:
    For a comparative study of the depletion of Kupffer cells by liposomal clodronate, liposomal propamidine and liposomal EDTA in the liver, see: Van Rooijen, N. & Sanders, A. 1996; Hepatology 23; 1239-1243.
  • Elimination & blocking of macrophages:
    For a review, see: Van Rooijen, N. & Sanders, A. 1997; J.Leuk.Biol. 62; 702-709.
  • Mechanism of Cell Death:
    For apoptosis of macrophages induced by liposomal clodronate 'in vitro', see: Van Rooijen, N., Sanders, A. & Van Den Berg, T. 1996; J.Immunol.Meth. 193; 93- 99.
  • Kupffer Cells in the Liver:
    For depletion of Kupffer cells in the rat liver, see: Van Rooijen, N. & Sanders, A. 1996; Hepatology 23; 1239-1243.
  • Ultrastructural demonstration of depletion of Kupffer Cells: For a demonstration of Kupffer cell depletion at an ultrastructural level, see: Van Rooijen, N., Van Nieuwmegen, R. & Kamperdijk, E.W.A. 1985; Virchows Arch. B (Cell Pathol.) 49, 375-383. 
  • Macrophages in the Spleen:
    MOUSE: For depletion and repopulation of macrophage subsets in the mouse spleen, see: Van Rooijen, N., Kors, N. & Kraal, G. 1989; J.Leuk.Biol. 45; 97-104. RAT: For depletion and repopulation of macrophage subsets in the rat spleen, see: Van Rooijen, N., Kors, N., Van Den Ende, M. & Dijkstra, C.D. 1990; Cell Tissue Res. 260, 215- 222.
  • Macrophages in Lymph Nodes:
    For depletion and repopulation of macrophages in lymph nodes, see: Delemarre, F.G.A., Kors, N., Kraal, G. & Van Rooijen, N. 1990; J.Leuk.Biol. 47; 251-257.

  • Alveolar Macrophages in Lung:
    For depletion of alveolar macrophages in the lung, see: Thepen, T., Van Rooijen, N. & Kraal, G. 1989; J.Exp.Med. 170; 499-509.

  • Peritoneal Macrophages:
    For depletion of peritoneal macrophages, see: Biewenga, J. et al. 1995; Cell Tissue Res.280; 189-196.

  • Testis Macrophages:
    For depletion of testis macrophages, see: Bergh, A., Damber, J-E. & Van Rooijen, N. 1993; J.Endocrinol. 136; 407-413.

  • Phagocytic Synovial Lining Cells in Joints:
    For depletion of phagocytic synovial lining cells in knee joints, see: Van Lent, P.L.E.M. et al. 1993;  Am.J.Pathol. 143; 1226-1237  & Van Lent, P.L.E.M. et al. 1996; Arthritis Rheum39; 1545-1555.

  • Perivascular and Meningeal Macrophages in the CNS:
    For depletion of perivascular and meningeal macrophages in the CNS, see: Polfliet, M.M.J. et al. 2001; J. Neuroimmunol. 116; 188-195, 2001.



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