Wednesday, January 4, 2012

Isolation of murine Kupffer cells

Method:
1) Sacrifice the mice and bleed them by aortic section.
2) Pin the mice-abdomen up- to the dissecting board.
3) Clean the mouse with ethanol. Work under a sterile hood.
4) Make a ventral midline incision in order to expose the peritoneal cavity.
5) Cut the vena cava to prevent reflux of blood and then perfuse the liver.
6) Perfuse the liver through the portal vein at a flow rate of 10 ml/min for 5 minutes with dissociation buffer at 37 C using a 24-gauge cannula.
7) Remove the liver from animal using a sterile forceps.
8) Mince the liver into small pieces and push the pieces through a sterile mesh using  the rubber end of a sterile syringe plunger and collect the cells in a sterile petri dish.
9) Transfer the cells to a 150 cc tissue culture flask.
10) Add 100 ml of a 4:1 ratio of GBSS dissociation buffer to the cells. (Dissociation buffer: 0.1 mM L-aspartic acid, 0.2 mM L-threonine, 0.3 mM L-serine, 0.5 mM glycine, 0.6 mM L-alanine, 0.9 mM L-glutamic acid, 3 mM KCl, 0.7 mM NaH2PO4.H2O, 0.5 mM MgCl2, 24 mM NaHCO3, 20 mM glucose, 20 mM fructose, 197 mM saccharose, 0.05% collagenase A pH 7.4)
11) Incubate with continuous agitation for 1 hour at 37 C, 5% CO2.
12) Filter the cells suspension through a sterile mesh into a sterile tube.
13) Centrifuge at 300 g for 15 minute at 4 C.
14) Resuspend the cell pellets in 10 ml medium.
15) Divide 10 ml of resuspended cell pellets to two 5 ml. Pour 5 ml of the cell suspension into two 15 ml centrifuge tubes.
16) Mix the cell suspension with 5 ml of 30% metrizamide in GBSS (Invitrogen) without NaCl.
17) Top the solution with 1 ml of GBSS (with NaCl).
18) Spin the cells at room temperature at 1400 g for 45 minutes in a table-top centrifuge with out brake.
19) Recover the cells from the interface of the step-gradient with a sterile pipette.
20) Purify the macrophages by centrifugal elutriation.

For more information about macrophage depletion visit www.clodrosome.com 





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