This article describes the isolation and purification of Kupffer cells from mouse liver. Kupffer cells are crucial for liver immunity and play a role in protecting against infections.
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.
To purify Kupffer cells, place one perfused liver in a Petri dish with 15 milliliters of Kupffer cell isolation medium. With scissors or tweezers, rupture the Glisson's capsule, and release all liver cells into the medium. Filter the solution through a 100-micrometer cell strainer into a centrifuge tube. Then, centrifuge the cell suspension at 50 g at 4 degrees Celsius for two minutes.
Parenchymal cells will be in the pellet, and non-parenchymal cells will be in the supernatant. Collect the supernatant in a clean centrifuge tube and centrifuge at 50 g for 2 minutes. Repeat for a total of 4 transfers and spins.
Next, centrifuge the supernatant at 1,350 g for 15 minutes to pellet the non-parenchymal cells. Discard the supernatant and resuspend the pellet in 10 milliliters of Kupffer cell isolation medium. Add the non-parenchymal cell solution to the discontinuous 25/50% isotonic gradient, previously prepared according to the text protocol, and centrifuge at 850 g for 15 minutes without acceleration or break.
Using a 10-milliliter serological pipette, aspirate about 12 milliliters of the enriched Kupffer fraction that appears turbid within the 25% SIP fraction close to the 25/50% SIP interface. Transfer the cells into a centrifuge tube containing 35 to 40 milliliters of Kupffer cell isolation medium, and gently mix. Then, centrifuge at 1,350 g and 4 degrees Celsius to pellet the cells.
Discard the supernatant and use 5 to 10 milliliters of pre-warmed medium to resuspend the cells. With a hemocytometer, count the cells and use Trypan blue staining to measure the viability. Plate the purified non-parenchymal cells in 24-well plates, at a density of 5 x 105 cells per well. Incubate the cells at 37 degrees Celsius and 5% carbon dioxide for 30 minutes. Then, gently remove the medium.
Use pre-warmed HBSS to wash the cells and replace it with 500 microliters per well of fresh pre-warmed Kupffer cell culture medium.
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