For analyses of epithelial cell phenotypes, luminal (EPCAMHi/CD49fLo) or non-luminal (EPCAMLoCD49fHi) and an epithelial stem cell phenotype (EPCAMLo/CD49fHi/CD24Lo/CD44Hi)8 (link), 48 , 49 (link), control or CXCL12γ overexpressing epithelial cells (PNT2, MCF10A) (1 x 105) were seeded onto 12-well culture plates and were cultured for 3 days. The cells were incubated with FITC anti-human CD326 (EPCAM) antibody (cat. 324204, Biolegend, San Diego, CA), PE/Cy7 anti-human CD49f antibody (cat. 313622, Biolegend), APC/Cy7 anti-human CD24 antibody (cat. 311132, Biolegend), APC anti-CD44 antibody (cat. 559942, BD Biosciences, San Jose, CA) for 30 min at 4°C. DAPI was used for determining cell viability. The cell phenotypes were evaluated by FACS analyses (FACSAria IIu flow-cytometer, Becton Dickinson, Mountain View, CA). Assays were performed in triplicate and the results are representative of three independent experiments.
Protocol detail
Characterization of Epithelial Cell Phenotypes
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Anti antibody
Antibody
Assays
Cd44
Cd49f
Cell
Cell viability
Dapi
Epcam
Epithelial cells
Fitc
Human
Luminal
Phenotypes
Stem
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Variable analysis
independent variables
- Overexpression of CXCL12γ in epithelial cells (PNT2, MCF10A)
- Epithelial cell phenotypes: luminal (EPCAM^Hi/CD49f^Lo), non-luminal (EPCAM^Lo/CD49f^Hi), and epithelial stem cell (EPCAM^Lo/CD49f^Hi/CD24^Lo/CD44^Hi)
- Control epithelial cells (PNT2, MCF10A) without CXCL12γ overexpression
- Cell seeding density (1 x 10^5 cells)
- Culture duration (3 days)
- Incubation with FITC anti-human CD326 (EPCAM) antibody, PE/Cy7 anti-human CD49f antibody, APC/Cy7 anti-human CD24 antibody, APC anti-CD44 antibody for 30 min at 4°C
- DAPI staining for cell viability determination
- Positive control: Not specified
- Negative control: Control epithelial cells (PNT2, MCF10A) without CXCL12γ overexpression
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