Project description:To validate the CD49f/CD61 flow cytometry panel for resolving the three major mammary epithelial lineages, mammary epithelial cells from MMTV-Cre mice were collected by FACS and analyzed by RNA-seq for comparison to previously published single-cell RNA-seq studies, which non-biasedly identified the three major lineages: basal, alveolar progenitor (AP), and hormone-sensing luminal (HSL) cells.
Project description:To delineate epithelial subpopulations in human mammary tissue, hematopoietic and endothelial cells were depleted from freshly isolated cell suspensions derived from reduction mammoplasties by fluorescence-activated cell sorting. The resultant Lin- population was fractionated into four distinct subpopulations using CD49f (α6-integrin) and epithelial cell adhesion molecule (EpCAM; also referred to as CD326 and ESA). Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as fibroblast-enriched stromal (CD49f -EpCAM-), mammary stem cell (MaSC)-enriched (CD49f hiEpCAM-), luminal progenitor (CD49f +EpCAM+), and mature luminal (CD49f –EpCAM+) cell subpopulations. Microarray profiling was used to derive gene expression signatures representative of these subpopulations using freshly sorted cells (>90% purity) from normal breast tissue. The four mammary cell subpopulations were found to have distinct gene expression profiles.
Project description:To delineate epithelial subpopulations in human mammary tissue, hematopoietic and endothelial cells were depleted from freshly isolated cell suspensions derived from reduction mammoplasties by fluorescence-activated cell sorting. The resultant Lin- population was fractionated into four distinct subpopulations using CD49f (α6-integrin) and epithelial cell adhesion molecule (EpCAM; also referred to as CD326 and ESA). Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as fibroblast-enriched stromal (CD49f -EpCAM-), mammary stem cell (MaSC)-enriched (CD49f hiEpCAM-), luminal progenitor (CD49f +EpCAM+), and mature luminal (CD49f âEpCAM+) cell subpopulations. Microarray profiling was used to derive gene expression signatures representative of these subpopulations using freshly sorted cells (>90% purity) from normal breast tissue. The four mammary cell subpopulations were found to have distinct gene expression profiles. Four mammary cell subpopulations from three individual patient samples were analyzed.
Project description:We defined CD49f-high, CD49f-low and CD49f-neg mesenchymal subpopulations in the dermis. Transcriptome analysis revealed that CD49fhigh cells highly express gene regulatory network of neural crest cells, while CD49flow cells were enriched with melanocyte lineage development and maintenance related genes. The identity and function of above cell populations were further verified by lineage tracing.
Project description:This experiment shows differential expression of genes in the luminal, basal and stromal subpopulations from SNAI2+/+ and SNAI2 LacZ/LacZ mammary epithelial cells. Luminal, basal and stromal populations were sorted from SNAI2+/+ and SNAI2 LacZ/LacZ mammary epithelial cells based on expression of CD49f and Epcam.
Project description:We have carried out microarray-based gene expression analysis of cell subpopulations isolated by flow cytometry, on the basis of staining with antibodies against CD45, CD49f, Sca-1 and the 33A10 antigen, from freshly prepared mouse mammary glands. RNA isolated from cell populations was co-hybridised with control mouse RNA on to an in-house (Breakthrough Breast Cancer Centre) cDNA mouse microarray containing 13825 features (NIA 15K Mouse cDNA clone set). LIMMA analysis was used to analyse data and determine changes in RNA expression levels.
Project description:The transgenic FVB/N 11.5kb-GFP mouse line was generated using the 11.5 kb s-SHIP promoter. During puberty, GFP expression in the mammary gland is specifically restricted to cap cells, which exhibit stem cell-like properties. To investigate the molecular basis underlying the differences between these basal GFP⁺ cap cells and other mammary epithelial subpopulations, we employed fluorescence-activated cell sorting to isolate four distinct mammary cell populations from 6-week-old mice, based on GFP expression in combination with CD24 and CD49f cell surface markers. These populations were subsequently analyzed for gene expression profiles.
Project description:The metabolic status of individual cells in microbial cultures can differ being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a ?aceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the ?aceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE.
Project description:To further characterize the molecular and biological features of the Sca-1+ luminal cells, we performed an RNA-seq analysis using FACS-isolated distinct cell lineages in adult mouse prostates, including the Lin-CD24lowCD49fhigh basal cells, Lin-CD24-CD49f- stromal cells, Lin-CD24highCD49flowSca-1- luminal cells, and Lin-CD24highCD49flowSca-1+ luminal cells.