OUTLINE

1. preparethecholisterolsolutioninaroundbottombulb.
2. add0.86mlofphosphatidilcholine.
3. mixwellandapplythevacuumevaporationsystemwithrotation.
4. thebulbshouldbeplacedonthe37ѓCwaterbath.
5. lettoevaporate.
6. aftercompleteevaporationturnoffthevacuumpompandcirculatingcoldwater.
7. addwhether10mlofPBSorwatersolutionofclodronateandapplyrotationtillthelipidfilmissolved,RT(!)
8. transferthesolutionintotheultracentrifugeplastictubeandapplyN2gasthroughtheBilbateЖPipetteplacedonthebottomofthetubefor60-90min,RT.
9. sonicatefor3min,50Wt(onice)
10. applyN2gasthroughtheBilbateЖpipetteplacedonthebottomofthetubefor60-90min,RT.
11. centrifugefor15minat10000rpm(rotorID=JA20),24ѓC
12. aspiratethesolutionundertheliposomes"ring(incaseofliposome-clodronate)oraspiratethesupernatant(SN)(forliposome-PBS)
13. resUSPendinPBS,10ml
14. centrifugefor30min,20000rpm,24ѓC
15. aspirateSN
16. resuspendinPBS,10ml
17. centrifugefor30min,20000rpm,24ѓC
18. resuspendinPBS,4ml(sterile!)
19. storeat4ѓCfornomorethan7-10days
20. inject200µl/mouse,i.v.

1. cholisterolsolution=cholisterol,8mg+chloroform,10ml
2. phosphatidilcholine(Sigma)=chloroformsolutionofphosphatidilcholine,100mg/ml
3. clodronatesolution(RT!)=clodronate,2.5g+ddH2O,10ml
4. PBS

1. VanRooijen,N.(1989).Theliposome-mediatedmacrophage"suicide"technique.JImmunolMethods124(1):1-6.
2. Jordan,M.B.,vanRooijen,N.,Izui,S.,Kappler,J.andMarrack,P.(2003).Liposomalclodronateasanovelagentfortreatingautoimmunehemolyticanemiainamousemodel.Blood101(2):594-601.
3. Alves-Rosa,F.,Stanganelli,C.,Cabrera,J.,vanRooijen,N.,Palermo,M.S.andIsturiz,M.A.(2000).Treatmentwithliposome-encapsulatedclodronateasanewstrategicapproachinthemanagementofimmunethrombocytopenicpurpurainamousemodel.Blood96(8):2834-40.
4. vanRooijen,N.,Sanders,A.andvandenBerg,T.K.(1996).Apoptosisofmacrophagesinducedbyliposome-mediatedintracellulardeliveryofclodronateandpropamidine.JImmunolMethods193(1):93-9.

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