Conditioned media (CMs) from human and mouse macrophages, i.e., M0 CM, M(IFN-γ)
CM, M(IFN-γ→IL-4) CM, M(IL-4) CM, and M(IL-4→IFN-γ) CM were collected and
centrifuged to remove cell debris. Then, CMs from above-mentioned groups were
incubated with primary mouse hepatocytes or human liver cell lines (HL-7702 and
HepG2) for 6 h, and then cell apoptosis was induced by human and mouse TNF-α (50
μg/ml, Peprotech)/d -GalN (100 mg/ml, Sigma-Aldrich) (GA for short) for
12 h.27,28 To evaluate hepatocyte apoptosis, primary mouse
hepatocytes and HL-7702/HepG2 cells were stained with rabbit anti-mouse cleaved
caspase-3 (Abcam, Cambridge, MA, USA) and FITC-conjugated goat anti-rabbit IgG
(eBioscience, San Diego, CA, USA). A Nikon Inverted Fluorescence Microscope
ECLIPSE Ti and NIS-Elements F3.0 Software (Nikon Corporation, Tokyo, Japan) was
applied for image capture. Image J software was used to quantify the expression
of cleaved caspase-3.
CM, M(IFN-γ→IL-4) CM, M(IL-4) CM, and M(IL-4→IFN-γ) CM were collected and
centrifuged to remove cell debris. Then, CMs from above-mentioned groups were
incubated with primary mouse hepatocytes or human liver cell lines (HL-7702 and
HepG2) for 6 h, and then cell apoptosis was induced by human and mouse TNF-α (50
μg/ml, Peprotech)/
12 h.27,28 To evaluate hepatocyte apoptosis, primary mouse
hepatocytes and HL-7702/HepG2 cells were stained with rabbit anti-mouse cleaved
caspase-3 (Abcam, Cambridge, MA, USA) and FITC-conjugated goat anti-rabbit IgG
(eBioscience, San Diego, CA, USA). A Nikon Inverted Fluorescence Microscope
ECLIPSE Ti and NIS-Elements F3.0 Software (Nikon Corporation, Tokyo, Japan) was
applied for image capture. Image J software was used to quantify the expression
of cleaved caspase-3.