Easysep mouse epithelial cell enrichment kit

Single cells were isolated from Gas6^-/- or wildtype mice (3 mice per group) and resuspended in HBSS containing 2% FBS and 100 mM Hepes (HBSS⁺) at a concentration of 1 × 10⁷ cells/ml. Antibody staining was performed as previously described [35 (link)], and antibodies are listed in S1 Table. Lineage exclusion was achieved with the EasySep Mouse Epithelial Cell Enrichment Kit (Stem Cell Technologies) prior to antibody staining. Cells were filtered with a 35 μm cell strainer, stained with 5 nM SYTOX (Life Technologies), and analyzed by flow cytometry using BD LSRII flow cytometer. Data were analyzed using FlowJo version 10.

Isolation of lung epithelial cells from C57BL/6 mouse was performed as described previously by using the EasySep™ mouse epithelial cell enrichment kit (Cat. no. 19758; STEMCELL Technologies, Canada) [4 (link),28 (link)–30 (link)]. Briefly, mouse lungs teased out of bronchiole, chopped into tiny pieces, were enzymatically homogenized, passed through a 40 μm cell strainer and then subjected to mouse epithelial cell isolation kit containing a cocktail of anti-CD31, anti-CD45, anti-TER119 antibodies following manufacturer’s protocol. Purity of the enriched lung cells were validated by staining with anti-B220, anti-CD3, anti-CD11b, anti-CD11c, anti-NK1.1 and F4/80 antibodies before (8.99%, 14.47%, 9.03%, 4.30%, 5.82% and 23.71% respectively) and after (0.28%, 0.53%, 1.09%, 1.15%, 0.15% and 1.48% respectively) enrichment. Recovery of PLE cells ranged from 4 X 10⁶ to 5 X 10⁶ with above 99% purity.
BAL cells were isolated by using a catheter-attached 1 ml syringe through a small transverse incision of the exposed trachea, as described before [4 (link)]. BAL cells were cultured overnight to enrich the adhering macrophage population, which was found to be 92.33% pure by F4/80 staining.

Fresh mammary glands and tumor tissues were cut into small pieces. Tissues were digested using a “Digestion I” solution (DMEM/F12 (Gibco, Life Technologies, #11330-032) supplemented with 5% fetal bovine serum (FBS) (Prime) (ExCell Bio, #FSP500), 5 µg/ml insulin (Sigma-Aldrich, #I1882), 500 ng/ml hydrocortisone (Sigma-Aldrich, #H0888), 10 ng/ml EGF(Invitrogen, Thermo Fisher Scientific, #13247-051), 300 U/ml collagenase III (Worthington, #S4M7602S), and 100 U/ml hyaluronidase (Sigma-Aldrich, #H3506) for 2 h at 37 °C and a “Digestion II” solution (HBSS (Gibco, Life Technologies, #14170), 5 mg/ml Dispase II (Roche Diagnostics, # 04942078001), and 0.1 mg/ml deoxyribonuclease (Worthington Biochemical, #LS002145)) for 5 min at 37 °C, followed by 0.25% trypsin-EDTA (Gibco, Life Technologies, #25200) treatment for 1 min and red blood cell lysis buffer (Invitrogen, Thermo Fisher Scientific, #00-4333-57) treatment for 5 min at room temperature. Samples were filtered through a 40 µm cell strainer to a 5 ml polystyrene tube. Epithelial cells were enriched using the EasySep Mouse Epithelial Cell Enrichment Kit (STEMCELL TECHNOLOGIES, #19868).