Kupffer Cell Isolation from Injured Liver

Kupffer cells were isolated 24 hour after injury as described by Kuriakose et al. (18 (link)) with minor modifications. Rinsed liver tissue was incubated at 37°C for 30 minutes in collagenase IV (5000 CDU/mL), run through a gentleMACS Dissociator (Miltenyi Biotec, Auburn, CA), and passed through a sterile 100 um strainer into phosphate-buffered saline (Miltenyi Biotec, Auburn, CA) containing 0.5% bovine serum albumin. The hepatocytes were removed by centrifugation at 20xg for 4 minutes, and the residual cell suspension was centrifuged twice at 300xg for 10 minutes at 4°C with the cell pellet washed with Red Blood Cell Lysis Solution between centrifugations. CD11b⁺ cells were enriched by positive selection using AUTOMACS (Miltenyi Biotec Auburn, CA). Enriched liver CD11b⁺ cells isolated by this method have been shown to be greater than 96% positive for F4/80 expression as assessed by flow cytometry (18 (link)).