2.6 Flow cytometry analysis
Isolated primary NK cells were co-cultured with TGF-β-treated HSCs at the ratio of 10:1 as previously described (Melhem et al., 2006). After attached, co-cultured cells were treated with CM of macrophages for 24 h and collected after digestion without EDTA and centrifuged at 2000 g for 5 minutes at 4°C. Fixed cells by paraformaldehyde were first incubated with α-SMA antibody (1 : 400) and 5 μl 7-AAD solution from 7-AAD staining kit (BD Biosciences, CA, USA). For α-SMA staining, cells were stained with 4μM Goat anti-Rabbit IgG (H+L) Secondary Antibody Alexa Fluor 488 for 30 min in the dark at room temperature and further stained with 7-AAD binding buffer offered by staining kit. Then the α-SMA/7-AAD signal were detected by the CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA), and the FlowJo software (v.10.3) was used for subsequent analysis of relative data.