Mammary Cell Immunophenotyping Protocol

Mouse mammary cells were preblocked with 10% normal rat serum and then incubated with the following primary antibodies: CD31-biotin (clone 390, eBioscience), CD45-biotin (clone 30-F11, eBioscience), Ter119-biotin (clone Ter119, eBioscience), BP-1-biotin (clone 6C3, eBioscience), EpCAM-APC (clone G8.8, BioLegend), CD49f-AF488 (clone GoH3, BioLegend), CD49b-PE (HMα2, BioLegend), and Sca1-PE/Cy7 (clone D7, BioLegend). In some experiments, cells were stained with CD24-PE (clone M1/69, BioLegend) and CD29 (clone HMβ1-1, BioLegend). CD45, Ter119, CD31, and BP-1 were used to deplete contaminating hematopoietic, endothelial, and a proportion of stromal cells, respectively (collectively termed Lin + cells). Biotin-conjugated antibodies were detected with Streptavidin-APC-Cy7 (BioLegend). Cells were then filtered through a 30-μm cell strainer and incubated with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) and were analyzed by using an LSRII (Becton Dickinson) and were sorted on a FACSAria I (Becton Dickinson). The flow-cytometry gating strategy was as previously described [11 (link)].

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Shehata M., van Amerongen R., Zeeman A.L., Giraddi R.R, & Stingl J. (2014). The influence of tamoxifen on normal mouse mammary gland homeostasis. Breast Cancer Research : BCR, 16, 411.

Publication 2014

CD31-biotin (clone 390, CD45-biotin (clone 30-F11, Ter119-biotin (clone Ter119, CD49b-PE (HMα2, CD29 (clone HMβ1-1, Streptavidin-APC-Cy7 4′,6-diamidino-2-phenylindole (DAPI; FACSAria I

Antibodies Biotin Cd49f Cells Clone Dapi Endothelial Epcam Flow cytometry Hematopoietic Mammary Mouse Sca1 Serum Streptavidin Stromal cells

Corresponding Organization :

Other organizations : University of Cambridge, University of Amsterdam

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Variable analysis

independent variables

Treatment with primary antibodies: CD31-biotin, CD45-biotin, Ter119-biotin, BP-1-biotin, EpCAM-APC, CD49f-AF488, CD49b-PE, Sca1-PE/Cy7, CD24-PE, CD29

dependent variables

Flow cytometry analysis and cell sorting

control variables

Preblocking with 10% normal rat serum
Depleting contaminating cells (CD45+, Ter119+, CD31+, BP-1+)
Filtering cells through a 30-μm cell strainer
Incubation with DAPI

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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