MUTCHLER产品目录_第1页_Chemicalbook
来自 : 蚂蚁淘
OUTLINEClodronate(dichloromethylenediphosphonate)isusedfortreatmentofosteolyticdiseasesandosteoporosisItseffectisduetoinhibittionofthefunctionofosteoclasts.Thisdrugisalsotakenupbymacrophagesandrapidlycausesapoptosisofthesecells.
PROTOCOL- preparethecholisterolsolutioninaroundbottombulb.
- add0.86mlofphosphatidilcholine.
- mixwellandapplythevacuumevaporationsystemwithrotation.
- thebulbshouldbeplacedonthe37ѓCwaterbath.
- lettoevaporate.
- aftercompleteevaporationturnoffthevacuumpompandcirculatingcoldwater.
- addwhether10mlofPBSorwatersolutionofclodronateandapplyrotationtillthelipidfilmissolved,RT(!)
- transferthesolutionintotheultracentrifugeplastictubeandapplyN2gasthroughtheBilbateЖPipetteplacedonthebottomofthetubefor60-90min,RT.
- sonicatefor3min,50Wt(onice)
- applyN2gasthroughtheBilbateЖpipetteplacedonthebottomofthetubefor60-90min,RT.
- centrifugefor15minat10000rpm(rotorID=JA20),24ѓC
- aspiratethesolutionundertheliposomes"ring(incaseofliposome-clodronate)oraspiratethesupernatant(SN)(forliposome-PBS)
- resUSPendinPBS,10ml
- centrifugefor30min,20000rpm,24ѓC
- aspirateSN
- resuspendinPBS,10ml
- centrifugefor30min,20000rpm,24ѓC
- resuspendinPBS,4ml(sterile!)
- storeat4ѓCfornomorethan7-10days
- inject200µl/mouse,i.v.
SOLUTIONS- cholisterolsolution=cholisterol,8mg+chloroform,10ml
- phosphatidilcholine(Sigma)=chloroformsolutionofphosphatidilcholine,100mg/ml
- clodronatesolution(RT!)=clodronate,2.5g+ddH2O,10ml
- PBS
ADDITIONALINFOIt"simportanttokeeptheclodronatewatersolutionandliposomes-clodronateatroomtemperature(RT)unlessotherspecified.
SCHEME
[enlarge]REFERENCES- VanRooijen,N.(1989).Theliposome-mediatedmacrophage"suicide"technique.JImmunolMethods124(1):1-6.
- Jordan,M.B.,vanRooijen,N.,Izui,S.,Kappler,J.andMarrack,P.(2003).Liposomalclodronateasanovelagentfortreatingautoimmunehemolyticanemiainamousemodel.Blood101(2):594-601.
- Alves-Rosa,F.,Stanganelli,C.,Cabrera,J.,vanRooijen,N.,Palermo,M.S.andIsturiz,M.A.(2000).Treatmentwithliposome-encapsulatedclodronateasanewstrategicapproachinthemanagementofimmunethrombocytopenicpurpurainamousemodel.Blood96(8):2834-40.
- vanRooijen,N.,Sanders,A.andvandenBerg,T.K.(1996).Apoptosisofmacrophagesinducedbyliposome-mediatedintracellulardeliveryofclodronateandpropamidine.JImmunolMethods193(1):93-9.
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