Isolation and Immunophenotyping of Mammary Epithelial Cells

MECs were lineage-depleted utilizing anti-CD31 (0.5mg/mL,102401; RRID:AB_312896), anti-TER-119 (0.5mg/mL,116201; RRID:AB_313702), and anti-CD45 (0.5mg/mL, 103102; RRID:AB_312967) antibodies (Biolegend) conjugated to sheep anti-rat IgG Dynabeads (Invitrogen, 11035). Lineage-depleted cells were stained with Ghost Violet 450 viability dye (TONBO Biosciences, 13–0863-T100). For immunotyping, MECs were incubated with CD16/32 antibodies (ThermoFisher; 14–0161-82; RRID:AB_467133), followed by fixable viability dye eFluor 780 (eBiosciences; 65–0865). Antibodies are listed in Table S1. Cells were analyzed using BD LSR Fortessa (BD Biosciences). Immune cells were identified as described (21 (link)). Gates were set on fluorescence minus one controls, and data were analyzed using FlowJo software (TreeStar V10; RRID:SCR_008520). Progenitor assays were conducted as described (5 (link)).

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Chamberlin T., Clack M., Silvers C., Kuziel G., Thompson V., Johnson H, & Arendt L.M. (2020). Targeting obesity-induced macrophages during preneoplastic growth promotes mammary epithelial stem/progenitor activity, DNA damage and tumor formation. Cancer research, 80(20), 4465-4475.

Publication 2020

anti-CD31 anti-TER-119 anti-CD45 sheep anti-rat IgG Dynabeads Ghost Violet 450 viability dye CD16/32 antibodies eFluor 780 BD LSR Fortessa

Anti igg Antibodies Assays Cells Fluorescence Ghost Sheep Violet

Corresponding Organization :

Other organizations : University of Wisconsin–Madison

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Variable analysis

independent variables

Lineage depletion using anti-CD31, anti-TER-119, and anti-CD45 antibodies

dependent variables

Viability of lineage-depleted cells
Immunophenotype of mammary epithelial cells (MECs)

control variables

Concentration of antibodies used for lineage depletion (0.5mg/mL)
Sheep anti-rat IgG Dynabeads used for lineage depletion
Ghost Violet 450 viability dye used for staining
CD16/32 antibodies used for immunotyping
Fixable viability dye eFluor 780 used for immunotyping
BD LSR Fortessa flow cytometer used for analysis
Fluorescence minus one controls used for gating
FlowJo software (TreeStar V10) used for data analysis
Progenitor assays conducted as described in reference 5

positive controls

Not explicitly mentioned

negative controls

Fluorescence minus one controls used for gating

Annotations

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