Cd95 apc

To identify the different B cell populations, two stains were performed in splenocytes from 4 to 5.5 month old mice (2 females and 1 male wt, and 4 females Kmt2d^−/−). First, to identify transitional, follicular and marginal zone populations, cells were stained with CD21-FITC (Biolegend, clone 7E9, #123407), CD5-PE (eBioscience, clone 53–7.3 #12-0051-81), CD23-PECY7 (Biolegend, clone B3B4 #101613), IgM-APC (Biolegend, clone RMM-1 #406509), and B220-Alexa700 (Biolegend, clone RA3 #103232). To identify intermediate plasma cells/plasmablast (IPC), plasma cells (PC) and germinal center populations, cells were stained with GL7-FITC (Biolegend, clone GL7 #144003), CD138-PE (Biolegend, clone 281-2 #142503), CD95-APC (eBioscience, clone 15A7 #17-0951-80), B220-Alexa700 (Biolegend, clone RA3 #103232). To determine the percentages of cell populations, values were normalized by % B220⁺ single live cells (single cells, 7-AAD negative. B220⁺). 7-AAD (Life Technologies) was used to identify dead cells. Data acquisition was performed in a BD LSR II Flow Cytometer (BD Biosciences) and analysis was performed with FlowJo software (Tree Star).

Enrichment for primary hepatocytes (PH) was performed by re-suspending the liver parenchymal fraction in HBSS/10%FBS at 10⁷ cells/ml, followed by incubation with CD95-Biotin conjugate (1:100, eBioscience) for 30 min at 4°C. Excess unbound primary antibody was removed via washing and centrifugation at 400 × g for 10 min at 4°C; the resulting cell pellet was resuspended in 90 μl of de-gassed MACS buffer/10⁷ total cells, followed by magnetic labeling with Anti-Biotin micro-beads (Miltenyi Biotec Inc., Auburn, CA) per the manufacturer's specifications. Magnetic separation was performed using an appropriate MACS column in the magnetic field of a VarioMACS separator, with CD95-expressing primary hepatocytes collected as eluate (Fig. 1B). Hepatocytes were assessed for cell surface marker expression by staining with the following rat anti-mouse antibody conjugates (Table S1): CD95-APC, EpCAM/CD326-PE-Cy7, CD45-eFluor 780, and c-Kit-PE (1:100; eBioscience). Single cell suspensions were washed at 50 × g and 4°C for 3 min, and resuspended in HBSS/10% FBS with 10 μg/ml PI, followed by flow cytometry using a FACSAria cell sorter.