Journal: The Journal of Experimental Medicine

Article Title: Hematopoietic stem and progenitor cells are present in healthy gingiva tissue

doi: 10.1084/jem.20200737

Figure Lengend Snippet: A unique monocyte population is present in the gingiva. (A) Gating strategy to identify monocytes in the gingiva and GI tract (numbers indicate frequencies expressed as mean ± SEM). (B) Quantification of monocytes as a percentage of live CD45 + Lin − CD11b + Ly6C +/− CD64 +/− cells (excludes Ly6C − CD64 − cells) in the gingiva of C57BL/6 and Balb/c mice and GI tract and skin of C57BL/6 mice. n = 6–13 mice per group. Lin = CD3ε, TCR-β, CD19, B220, NK1.1, Ter119, Siglec F, and Ly6G. (C and D) Representative staining of gingiva tissue for Ly6G + , Ly6C bright , and F4/80 + cells. (C) Images show locations of cells staining positive for each marker. (D) Representative sections stained for Ly6G, Ly6C, and F4/80. Solid white line indicates edge of tooth. Scale bar = 200 µm. Staining from three separate experiments with n = 2–3 per experiment. (E) Representative histograms showing staining for CD44, CD68, CCR2, and CX3CR1 by gingival Ly6C hi monocytes (Mo). Data from seven experiments with two to three mice per experiment. FMO, fluorescence minus one. (F) Cytospins of sorted gingival Ly6C hi monocytes and macrophages (Mϕ) stained with H&E. Scale bar = 10 µm. Images are representative of two independent experiments. (G) Representative FACS plots of sorted BM and gingiva monocytes that were cultured with M-CSF for 7 d and analyzed by FACS. Data from two independent experiments. (H) Monocytes were FACS purified from the blood, BM, gingiva, skin, and GI tract and analyzed by RNA-seq. n = 2–3 biological replicates per group. Heatmap of the expression profile of canonical monocyte and macrophage-associated genes. FPKM, fragments per kilobase per million mapped reads. (I) Representative FACS plots showing host- and donor-derived Ly6C hi monocytes in the blood and gingiva of head-shielded chimeras 20 wk after reconstitution. Numbers indicate percentage of cells in the gate. (J) Quantification of donor-derived (white bar) and host-derived (black bar) Ly6C hi gingival monocytes 12 and 20 wk after reconstitution in head-shielded chimeras. n = 6–11 mice per group from two to three experiments. (K) Chimerism of Ly6C hi monocytes in the lungs of torso-shielded chimeras (left) and GI tract of abdomen-shielded chimeras (right). The frequency of donor-derived Ly6C hi gingiva monocytes was normalized to that of blood Ly6C hi monocytes to determine percent chimerism. Data from one to two experiments with n = 3–9 mice per group. (L) Proportions of innate cells presented as the percentage of all CD45 + cells in the gingiva, GI tract, and skin. n = 3 from three independent experiments. Asterisks indicate significant differences compared with gingiva. (M) tSNE map of CD45 + Lin − Ly6G + gingival cells that were subjected to dimensional reduction based on Sca-1, cKit, CXCR2, CD45, Ly6G, CXCR4, MHCII, CD11b, Ly6C, CD11c, CD101, and Lin. Identified subpopulations are highlighted in the tSNE plot. Data representative of two experiments with n = 2–3 mice. Data are presented as mean ± SEM. Statistical comparisons were performed using a one-way ANOVA with a post hoc Tukey’s test (B) and a two-way ANOVA with a Tukey’s (L) and Holm–Šídák post hoc test (J); ****, P < 0.0001; **, P < 0.01; *, P < 0.05.

Article Snippet: Where appropriate, dimensional reduction was performed using the t-distributed stochastic neighbor embedding (tSNE) algorithm in FlowJo.

Techniques: Staining, Marker, Fluorescence, Cell Culture, Purification, RNA Sequencing Assay, Expressing, Derivative Assay

Journal: Experimental hematology

Article Title: Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts

doi: 10.1016/j.exphem.2021.02.012

Figure Lengend Snippet: Hypoxia promotes the development of megakaryoerythroid progenitors. (A) Schematic of myeloerythroid differentiation from hematopoietic stem cells. The surface markers used for immunophenotyping of the cells interrogated in this study are indicated. (B) Percentage of multipotent progenitors (MPP), common myeloid progenitors (CMP), megakaryoerythroid progenitors (MEP), and granulocyte−monocyte progenitors (GMP) in cultures incubated in hypoxia and normoxia. For MPPs, the percentage of positive cells in the Lin − /Live population was determined, and for CMPs, GMPs, and MEPs, the percentage of positive cells in the MPP population was determined on days 1, 7, 14, and 21. Data are represented as the mean with standard error ( n = 4). Statistical analysis was performed using the Mann-Whitney test, and p values ≤ 0.05 were considered significant. (C) tSNE analysis was performed on flow cytometry standard files. tSNE plots for day 21 analysis revealing CD34 + and CD38 + cells in normoxia and hypoxia are represented in the Lin − /Live population. (D) tSNE analysis plots revealing the distribution of CMPs, GMPs, and MEPs in the CD34 + /CD38 + population in normoxia or hypoxia on day 21 are represented. (E) Histograms for CD45Ra and CD123 expression indicating relative distribution of CMPs (CD45Ra − /CD123 lo ), GMPs (CD45Ra + /CD123 lo ), and MEPs (CD45Ra − /CD123 − ) in normoxia and hypoxia. * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: t-Distributed stochastic neighbor embedding (tSNE) analysis was performed using FlowJo software (TreeStar) with default parameters (iterations = 1000, perplexity = 30).

Techniques: Incubation, MANN-WHITNEY, Flow Cytometry, Expressing

Journal: Experimental hematology

Article Title: Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts

doi: 10.1016/j.exphem.2021.02.012

Figure Lengend Snippet: Hypoxia enhances expression of erythroid markers. (A) Longitudinal analysis of CD71 relative to the erythroid marker CD235a in cultures incubated in normoxia or hypoxia. Percentages of CD71 + /CD235a − , CD71 − /CD235a + , and CD71 + /CD235a + in the CD34 − /Live population are illustrated. (B) Longitudinal analysis of CD71 relative to the erythroid marker CD239 in cultures incubated in normoxia or hypoxia. Percentages of CD71 + /CD239 − , CD71 − /CD239 + , and CD71 + /CD239 + cells in the CD34 − /Live population are illustrated. Data are represented as the mean with standard error ( n = 4). Statistical analysis was performed using the Mann-Whitney test, and p values ≤ 0.05 were considered significant. (C) tSNE plots revealing distribution of CD71 + , CD235a + , and CD239 + cells on day 21 in normoxia or hypoxia. (D) tSNE plots of overlay of CD71 + , CD235a + , and CD239 + cells in normoxia or hypoxia on day 21. tSNE analysis was performed in FlowJo. * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: t-Distributed stochastic neighbor embedding (tSNE) analysis was performed using FlowJo software (TreeStar) with default parameters (iterations = 1000, perplexity = 30).

Techniques: Expressing, Marker, Incubation, MANN-WHITNEY

Journal: Experimental hematology

Article Title: Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts

doi: 10.1016/j.exphem.2021.02.012

Figure Lengend Snippet: Expression of CD105 is persistent in hypoxia. (A) Longitudinal analysis of CD71 and CD105 in cultures incubated in normoxia or hypoxia. Percentages of CD71 + /CD105 − , CD71 − /CD105 + , and CD71 + /CD105 + in the CD34 − /Live population are illustrated. (B) Longitudinal analysis of CD105 relative to the erythroid marker CD235a in cultures incubated in normoxia or hypoxia. Percentages of CD105 + /CD235a − , CD105 − /CD235a + , and CD105 + /CD235s + cells in the CD34 − /Live population are illustrated. Data are represented as the mean with standard error ( n = 4). Statistical significance was calculated using the Mann-Whitney test, and p values ≤ 0.05 were considered significant. (C) tSNE plots revealing the distribution of CD105 + cells and overlay of CD71 + , CD105 + , and CD235a + cells on day 21 in normoxia or hypoxia. tSNE analysis was performed in FlowJo. (D) Longitudinal analysis of the CD49d and CD233 in cultures incubated in normoxia or hypoxia. Percentages of CD49d − /CD233 − , CD49d + /CD233 − , CD49d + /CD233 + , and CD49d + /CD233 − cells in the CD235a + population are illustrated. * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: t-Distributed stochastic neighbor embedding (tSNE) analysis was performed using FlowJo software (TreeStar) with default parameters (iterations = 1000, perplexity = 30).

Techniques: Expressing, Incubation, Marker, MANN-WHITNEY

Journal: Gastroenterology

Article Title: Combination of PD1 Inhibitor and OX40 Agonist Induces Tumor Rejection and Immune Memory in Mouse Models of Pancreatic Cancer

doi: 10.1053/j.gastro.2020.03.018

Figure Lengend Snippet: (A-E) tSNE plots of all T cells (A) with expression levels of CD4 (B), CD8 (C), NK1.1 (D), and PD-L1 (E) from all samples with number- and color-coded clusters.

Article Snippet: MATLAB for t-distributed stochastic neighbor embedding (tSNE) plot, heat map, and plot cluster tSNE.

Techniques: Expressing