Cd31 biotin

Thoracic and inguinal mammary glands were dissected from 9-week-old p38α^(lox/lox);MMTV-Cre and MMTV-Cre virgin females or 6-week-old p38α^(lox/lox);MMTV-Cre;PyMT and MMTV-Cre;PyMT female mice. Cell suspensions were prepared as previously described (Prater et al., 2013 (link)). FACS sorting was performed with a FACSAria sorter (BD Bioscience). Data were analyzed with the FlowJo software package. The antibodies used for FACS were from Miltenyi Biotec: Epcam-APC (#130102234), Epcam-FITC (#130102214), CD49f-APC (#130097250), CD49f-PE (#130097246), BP1-biotin (#130101844), CD31-biotin (#130101955), CD45-biotin (#130101952), and Ter119-biotin (#130101882); and from Biolegend: CD61-AF480 (#104311), Sca1-PECy7 (#108114), CD49b-PE (#103506), and Streptavidin-APCCy7 (#405208). FACS gating was based on single color staining and fluorescence-minus-one controls, and was checked with isotype controls (Prater et al., 2013 (link)). Gating strategies were as described previously (Prater et al., 2013 (link), Shehata et al., 2012 (link)). For colony-formation assays, freshly sorted cells were embedded in Matrigel (BD Pharmingen) as described previously (Shackleton et al., 2006 (link)).

Mammary glands of CO (vehicle)-treated and 4OHT-treated 8- to 9-week-old virgin littermate female FVB/Tfap2c^fl/fl/Krt5-Cre-ER^T2 mice were digested in a mixture of gentle collagenase/hyaluronidase (STEMCELL, cat. no. 07919) with 5% fetal bovine serum (FBS) in complete EpiCult-B (STEMCELL, cat. no. 05611) media for 15  h at 37°C. The red blood cells were lysed in NH₄Cl. The cell suspension was further digested in 0.25% trypsin-EDTA for 1 min then 5 U/mL dispase (STEMCELL, cat. no. 07913) plus 0.1 mg/mL DNase I (STEMCELL, cat. no. 07900) for 1 min followed by filtration through a 40-μm cell strainer. After washing in Hank’s balanced salt solution, cell viability was assessed by trypan blue. Single-cell isolation procedure was performed as described previously (Borcherding et al., 2015 (link)). FACS was carried out using FACS Aria (Becton Dickinson). The Lin^– population was defined as TER119^–, CD31⁻, and CD45^–. FACS data were analyzed using FlowJo software (v.10.1r7, Tree Star). The following antibodies were used: CD31-Biotin (BioLegend, 102404, 1:250), TER-119-Biotin (BioLegend, 116204, 1:250), CD45-Biotin (BioLegend, 103014, 1:250), Strep-PE-Cy7 (Invitrogen, 25-4317-82, 1:250), CD24-PE (BioLegend, 101807, 1:250), CD49f-FITC (BioLegend, 313606, 1:200).