Isolation and Culture of Mouse Kupffer Cells
Transkript
Kupffer cells are resident macrophages localized in the liver sinusoids that help protect the liver from infections.
To isolate mouse Kupffer cells, take a freshly harvested perfused mouse liver in a medium-containing dish. The liver perfusion step with collagenase digests the extracellular matrix, causing liver cell dissociation.
Rupture the liver capsule to release the dissociated liver cells, including parenchymal cells, PCs, and non-parenchymal cells, NPCs, including the Kupffer cells, endothelial cells, stellate cells, and the remaining erythrocytes.
Filter to remove debris. Perform low-speed differential centrifugation on the filtrate to selectively sediment the PCs. Collect the NPC-containing supernatant. Centrifuge to concentrate the cells.
Resuspend the cells in medium and layer on a discontinuous isotonic density gradient with two gradient media layers of differing densities. With centrifugation, the cells separate based on their density relative to the gradient medium.
Collect the enriched NPC layer at the gradient layer interphase. Transfer to a medium-containing tube and centrifuge. Seed the resuspended NPCs into a multi-well plate.
Within minutes, the Kupffer cells in suspension adhere readily to the plastic well surface, while other NPCs remain in the medium. Replace the medium with fresh medium to support the Kupffer cell growth.
