Macrophage Depletion by Clodronate

OUTLINE

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 37ѓC 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 Ж 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 Ж pipette placed on the bottom of the tube for 60-90 min, RT.
11. centrifuge for 15 min at 10000 rpm (rotor ID=JA20), 24ѓC
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, 24ѓC
15. aspirate SN
16. resuspend in PBS, 10 ml
17. centrifuge for 30 min, 20000 rpm, 24ѓC
18. resuspend in PBS, 4ml (sterile!)
19. store at 4ѓC 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