Isolation and Culture of Mouse Kupffer Cells
Transcrição
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.
