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.