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v i r a l   i m m u n i t y   &   p a t h o g e n e s i s   g r o u p

::  p r o t o c o l s  ::  m a c r o p h a g e  d e p l e t i o n  by  c l o d r o n a t e  ::


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OUTLINE

Clodronate (dichloromethylene diphosphonate) is used for treatment of osteolytic diseases and osteoporosis Its effect is due to inhibittion of the function of osteoclasts. This drug is also taken up by macrophages and rapidly causes apoptosis of these cells.

PROTOCOL
  1. prepare the cholisterol solution in a round bottom bulb.
  2. add 0.86 ml of phosphatidilcholine.
  3. mix well and apply the vacuum evaporation system with rotation.
  4. the bulb should be placed on the 37ƒC water bath.
  5. let to evaporate.
  6. after complete evaporation turn off the vacuum pomp and circulating cold water.
  7. add whether 10 ml of PBS or water solution of clodronate and apply rotation till the lipid film is solved, RT (!)
  8. transfer the solution into the ultracentrifuge plastic tube and apply N2 gas through the Bilbate Æ pipette placed on the bottom of the tube for 60-90 min, RT.
  9. sonicate for 3 min, 50 Wt (on ice)
  10. apply N2 gas through the Bilbate Æ pipette placed on the bottom of the tube for 60-90 min, RT.
  11. centrifuge for 15 min at 10000 rpm (rotor ID=JA20), 24ƒC
  12. aspirate the solution under the liposomes' ring (in case of liposome-clodronate) or aspirate the supernatant (SN) (for liposome-PBS)
  13. resuspend in PBS, 10 ml
  14. centrifuge for 30 min, 20000 rpm, 24ƒC
  15. aspirate SN
  16. resuspend in PBS, 10 ml
  17. centrifuge for 30 min, 20000 rpm, 24ƒC
  18. resuspend in PBS, 4ml (sterile!)
  19. store at 4ƒC for no more than 7-10 days
  20. inject 200 µl /mouse, i.v.
SOLUTIONS
  1. cholisterol solution = cholisterol, 8 mg + chloroform, 10 ml
  2. phosphatidilcholine (Sigma) = chloroform solution of phosphatidilcholine, 100 mg/ml
  3. clodronate solution (RT!) = clodronate, 2.5 g +  ddH2O, 10 ml
  4. PBS
ADDITIONAL INFO
It's important to keep the clodronate water solution and liposomes-clodronate at room temperature (RT) unless other specified.

SCHEME
clodronate-liposomes
[  enlarge  ]

REFERENCES
  1. Van Rooijen, N. (1989). The liposome-mediated macrophage 'suicide' technique. J Immunol Methods 124(1): 1-6.
  2. Jordan, M.B., van Rooijen, N., Izui, S., Kappler, J. and Marrack, P. (2003). Liposomal clodronate as a novel agent for treating autoimmune hemolytic anemia in a mouse model. Blood 101(2): 594-601.
  3. Alves-Rosa, F., Stanganelli, C., Cabrera, J., van Rooijen, N., Palermo, M.S. and Isturiz, M.A. (2000). Treatment with liposome-encapsulated clodronate as a new strategic approach in the management of immune thrombocytopenic purpura in a mouse model. Blood 96(8): 2834-40.
  4. van Rooijen, N., Sanders, A. and van den Berg, T.K. (1996). Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. J Immunol Methods 193(1): 93-9.
created by Andrei Musaji          ::          updated : 2004