Isolation of mouse primary intestinal epithelial cells (IECs) was
performed as described previously (Atarashi
et al., 2011 ). Briefly, 6-week-old C57BL/6 mouse intestines were
opened longitudinally, washed in phosphate-buffered saline (PBS) and cut
into 5-mm fragments. The epithelial integrity was disrupted by treatment
with 1 mM dithiothreitol (DTT) on a shaker. Liberated IECs were collected
and separated by Percoll gradient (Sigma Aldrich). Interface cells were
collected and used as IECs. Purified IECs were cultured in
high-glucose-formulated DMEM, supplemented with 10% FBS, 4 mM glutamine, 20
mM HEPES, 1 mM sodium pyruvate, and 100 U/mL penicillin/streptomycin. The
purity of isolated IEC was confirmed using FACS analysis with antibodies
against IEC markers, PE-anti-E-Cadherin antibody (Catalog: 147304,
BioLegend) and FITC-anti-Cytokeratin 18 antibody (Catalog: MA1-10326,
ThermoFisher Scientific). Isolated IEC purity and survival rate were both
> 94%.
performed as described previously (
et al., 2011
opened longitudinally, washed in phosphate-buffered saline (PBS) and cut
into 5-mm fragments. The epithelial integrity was disrupted by treatment
with 1 mM dithiothreitol (DTT) on a shaker. Liberated IECs were collected
and separated by Percoll gradient (Sigma Aldrich). Interface cells were
collected and used as IECs. Purified IECs were cultured in
high-glucose-formulated DMEM, supplemented with 10% FBS, 4 mM glutamine, 20
mM HEPES, 1 mM sodium pyruvate, and 100 U/mL penicillin/streptomycin. The
purity of isolated IEC was confirmed using FACS analysis with antibodies
against IEC markers, PE-anti-E-Cadherin antibody (Catalog: 147304,
BioLegend) and FITC-anti-Cytokeratin 18 antibody (Catalog: MA1-10326,
ThermoFisher Scientific). Isolated IEC purity and survival rate were both
> 94%.