Comprehensive Immune Cell Profiling in Liver and Blood

Kupffer cells were washed with PBS/1% BSA/0.1% NA azide (staining buffer), blocked with Fc block (BDbiosciences, San Jose, CA) for 10 min on ice, incubated with anti-human CD45 (BDbiosciences) for 30 min, followed by intra-cellular staining with anti-human CD68 (clone# Ki-M7, AbD serotec, Raleigh, NC) using BD Cytofix/Cytoperm fixation/permeabilization kits (BDbiosciences). Hepatocytes were washed with staining buffer followed by intra-cellular staining with goat anti-human serum albumin (MyBiosource, San Diego, CA). Approximately 5 million events were acquired for each staining. Peripheral blood (1–2 μl) drawn from the vein of the tail was collected in heparin-coated capillaries (Sigma-Aldrich, St Louis, MO), washed twice with PBS, suspended in PBS/1% BSA, blocked with Fc block for 15 minutes at RT and stained with anti-human CD235 (BDbiosciences) and Retic-Count (BDbiosciences) for 30 minutes. Bone marrow cells were obtained by flushing both tibias as described [21 (link)] and cell surface stained with anti-human CD45, anti-human CD71, anti-human CD235, and anti-mouse Ter-119 antibodies (BDbiosciences). Apoptosis in bone marrow cells was measured using Annexin V/7-ADD apoptosis detection kit (BD Biosciences).