Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 1. Maternal cells in breast milk were identified by protein, gene, and transcriptome analyses. Mature-stage breast milk from healthy donors was assessed for expression of cell marker proteins (e.g., EpCAM and CD45) in freshly isolated samples by flow cytometry. Then, RNA was isolated and used to conduct complementary gene expression analysis by RT-qPCR. Single-cell transcriptomics added depth to the analysis and defined the lactocyte population in breast milk.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Single-cell Transcriptomics

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 2. Mature-stage breast milk from donors contained epithelial lactocytes and a smaller population of immune cells. (A) Flow cytometry quantified expression of cell-specific markers: EpCAM+ (epithelial cells), CD45+ (immune cells), CK18+ (lactocytes), and VIM+ (mesenchymal cells). These data suggest that the CK18 antibody bounds poorly to lactocytes and likely underestimates true lactocyte percentages. FITC, fluorescein isothiocyanate. (B) The relative cellular composition in breast milk was affected by the health status of the mother and infant. Representative density plots are shown of EpCAM versus CD45 cell populations in milk from healthy donors (n = 10), donors with sick infants (n = 1), or donors with recent mastitis (n = 3). (C) Flow cytometry analysis of protein markers in all health statuses (n = 14) and the breakdown of (D) CD45+ immune cells and (E) VIM+ mesenchymal cells in different health statuses. (F) mRNA expression of gene markers corresponding to proteins in (C) indicates the presence of epithelial lactocytes (n = 6 to 7). Data are shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Flow Cytometry, Expressing, Comparison

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 3. Stem-like transcription factors were expressed in 5% of breast milk cells. Four stem-like transcription factors were assessed in breast milk epithelial cells. (A) From flow cytometry analysis, representative histograms of SOX2+, TRA-1-60+, NANOG+, and SSEA4+ stained and unstained samples are shown. (B) Flow cytometry analysis of EpCAM+ cells (n = 15) indicated high expression of SOX2 and TRA-1-60 with moderate to low expression of NANOG and SSEA4, respectively. (C) mRNA expression relative to a housekeeping gene was quantified for the genes corresponding to the proteins in (B) (n = 7). (D) Flow cytometry analysis of EpCAM+ cells and their increasing stemness based on positive expression of multiple stem-like markers (n = 15). Data are means ± SEM. One-way ANOVA with Tukey’s multiple comparison test; *P < 0.05; ****P < 0.0001.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Flow Cytometry, Staining, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Reduced Dendritic Cells Expressing CD200R1 in Children with Inflammatory Bowel Disease: Correlation with Th17 and Regulatory T Cells

doi: 10.3390/ijms161226143

Figure Lengend Snippet: Representative flow cytometry data to illustrate the gating strategy for FACS analysis of DC subsets. ( A ) Dendritic cell HLA-DR + Lin − cells were gated ( R1 ), and myeloid CD11c + mDCs ( R2 ), plasmacytoid CD123 + pDCs ( R3 ) were determined as a percentage of the total PBMC ( B ). Dot plots to illustrate the expression of CD200 and CD200R1 on the myeloid dendritic cell (mDC) subset of healthy controls and IBD patients ( C , D ) and on and plasmacytoid DCs (pDCs) of healthy controls and IBD patients ( E , F ).

Article Snippet: For regulatory T cell staining, 1 × 10 6 cells were resuspended in 100 μL flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA).

Techniques: Flow Cytometry, Expressing