Isolation of colonic macrophages from mice
Colonic macrophages were prepared as previously described (Zhao et al.,
2019). First, the whole colons of mice in each group were collected and
washed several times with HBSS. Then, the colonic tissue was cut into
small pieces of 0.5cm and added to predigestion solution (containing 1mM
DTT, 5mM EDTA, and 5% FBS) to remove epithelial cells and mucus. Then
centrifuge at 1200rpm for 5min. The supernatant was aspirated and the
remaining colon fragments were cultured with 8 times the volume of
digestive fluid containing 1mg/ml collagenase VIII, 0.2mg/ml DNaseI,
1mg/ml DispaseII, and 10% FBS. It was digested at 37°C for 90 minutes.
The samples were filtered, and
resuspended
in 40% and 80% fractions of the Percoll solution. After centrifugation
at 2000rpm for 20 min, live cells in the middle layer were collected and
the colonic macrophages were classified using Anti-F4/80 MicroBeads
UltraPure, mouse (#130-110-443, Miltenyi Biotec).