Goat anti mouse af647

Cells were stained with cell surface antibodies against the following antigens: CXCR4 (PE-conjugated; Life Technologies, MHCXCR404), cKit (APC-conjugated; Life Technologies, CD11705). For intracellular staining, cells were fixed with 1.6% paraformaldehyde at 37°C for 20 min, then permeabilized in saponin buffer (BioLegend). Intracellular antigens were probed with the following antibodies: AAT (Santa Cruz, sc-59438), AFP (Abcam, ab169552), 2C1 (a kind gift from David Lomas and Elena Miranda), and/or BiP (Invitrogen, PA1-014A), then incubated with goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-003) and donkey anti-rabbit AF488 (Jackson ImmunoResearch, 711-545-152). To assess proliferation by EdU incorporation, cells were incubated for 24 h with 10 μM EdU, then stained using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies, C10635) as per manufacturer’s instructions. For all flow cytometry experiments, gating was based on isotype-stained controls. Stained cells were quantified using a Stratedigm S1000EON, and data were analyzed using FlowJo (Tree Star).