2.7 Cell isolation, culture, and treatment
RAW cells (macrophage cell line) and LX-2 cells (HSC cell line) were cultured in DMEM medium (10% FBS) and incubated at 37°C with 5% CO2. For the preparation of conditional medium, cellular RNA and protein, RAW cells were treated with LPS (50 ng/mL), IFN-γ (2.5 ng/mL) and/or PIC Ⅱ (5, 10, 20 mM) for 24 h and further collected for RNA or protein extraction. The medium derived from above macrophages was then collected for protein extraction and subsequently used as conditioned medium (conditional medium of macrophages, named as CM-M) for establishing the co-culture systems of HSCs, NK cells, and neutrophils subsequently. The primary NK cells were isolated from mouse spleen using the NK extraction kit (P9310) purchased from Solarbio science & technology Co., Ltd (Beijing, China). The cells were pseudo-suspension cultured for about 6 h (Fehniger et al., 1997; Sliz et al., 2019), and then treated with PBS or different groups of CM-Ms. The cells were collected after 24 h of culture for subsequent experiments. For the co-culture of NK cells and LX-2 cells, HSCs (1 × 105 cells per well of a 6-well plate) were pre-treated by the fibroblast inducible factor TGF-β for 24 h, then the isolated NK cells (1 ×  106 cells per well of a 6-well plate), which has been pre-activated by IL-2 or CM-M for 6 h, were added at a ratio of 10:1 to establish the co-culture system for 24 h (Melhem et al., 2006) (Fig. 5A ). Then the cells and treat medium were collected for subsequent tests. The primary neutrophils were isolated from mouse bone marrow using a neutrophil extraction kit (P8550-200ml) purchased from Solarbio likewise. The cells were pseudo-suspension cultured for about 4 h, then treated with LPS (50 ng/mL) and/or PIC Ⅱ (5, 10 and 20 mM). After treatment, cell samples were further collected for subsequent immunofluorescence experiments or RNA extraction. For the co-culture of neutrophils and HSCs, activated HSCs (pre-treated by TGF-β) were co-cultured with isolated neutrophils at a ratio of 1 : 3 with the presence of different dose of PIC Ⅱ for 24 h (Casini et al., 1997; Zhou et al., 2018). After 24 h, cell samples were collected for extraction of cellular proteins, RNA or immunofluorescence analysis.