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.