Pe rat anti human cd184

For cell surface staining, liver cells were first incubated with DPBS-bovine serum albumin (BSA) 1.5% for 20 minutes at 4°C to prevent nonspecific binding. Next, the cells were washed with DPBS-BSA 1.5% and stained with 5 μL of PE rat anti-human CD184, APC mouse anti-human CD90, or their respective isotypes (BD Biosciences) for 30 minutes on ice. Finally, the cells were washed and fixed using a stabilizing fixative (BD Biosciences). For intracellular staining, liver cells were fixed and permeabilized with 200 μL of cytofix/cytoperm buffer (BD Biosciences) for 20 minutes at 4°C. The cells were then washed with perm/wash buffer and stained with PE rat anti-human CD184 or its isotype diluted in perm/wash for 30 minutes on ice. Next, the cells were washed twice and fixed with stabilizing fixative (BD Biosciences). Fluorescence was measured with a BD FACSCanto II cytometer on 10,000 cells using the FACSDiva software. Data analyses were performed with FlowJo software.

ADHLSCs were plated at passage 4 on 8-chamber slides (BD Biosciences) at a density of 5,000 cell/cm². Upon reaching 70% confluence, they were fixed with 4% paraformaldehyde for 15 minutes. After 2 washes with DPBS, ADHLSCs were blocked with DPBS-BSA 5% for 1 hour. Some of the samples were permeabilized with Triton 0.1% DPBS-BSA 1.5% buffer (BD Biosciences) for 20 minutes at 4°C. The cells were then stained with a PE rat anti-human CD184 for 2 hours at 4°C (BD Biosciences) and rinsed 3 times with DPBS. Finally, samples were embedded in ProLong Gold with DAPI (BD biosciences). Pictures were taken with an Axio Imager + ApoTome (Zeiss) at a 20x objective and analyzed with AxioVision software.