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: bioRxiv

Article Title: HIV-1 controllers possess a unique CD8+ T-cell activation phenotype and loss of control is associated with increased expression of exhaustion markers

doi: 10.1101/2024.04.09.588737

Figure Lengend Snippet: Flow cytometry was performed on PBMC from normal donors, HVL, LVL and LVL post control using the Zombie Yellow live/dead stain and antibodies for CD3, TIM3, CD38, TIGIT, PD1, CTLA4, HLA-DR, LAG3, and CD8. A) Cells identified as live (Zombie Yellow-) T-cells (CD3+) were clustered using the FlowSOM plugin in FlowJo. B) tSNE analysis and plotting was performed on the same live T-cell populations using the associated function in FlowJo. Final plot was colored according to the clusters identified in panel A. Cells from human subjects corresponding to C) HIV negative (ND), D) HVL, E) LVL, F) LVL post control, and G) HVL on ART were mapped back onto the tSNE plot. Circled populations are those unique to either LVLs, LVLs post control, or HVLs on ART and are labelled with an identifying number that corresponds to the cluster (A) of which they are a sub-population.

Article Snippet: Clustering and t-Distributed Stochastic Neighbor Embedding (tSNE) dimensionality reduction of flow cytometry data was performed using the FlowSOM and tSNE functions available within FlowJo v10.

Techniques: Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Comparative analysis of human gut- and blood-derived mononuclear cells: contrasts in function and phenotype

doi: 10.3389/fimmu.2024.1336480

Figure Lengend Snippet: FItSNE projection of the various cytokine-expressing cell subtypes in PBMC and RMC. Data from healthy donor PBMC (n=8) or RMC (n=11) samples, obtained via flow cytometry, were concatenated to generate a single FItSNE projection of the different unbiased cell clusters based on their cytokine and markers expression, calculated with FlowSOM. The FItSNE projection along with the percentage of events of each cell cluster for both PBMC (A, B) and RMC (C, D) are shown. FItSNE analysis was performed with the tSNE function in FlowJo, FItSNE algorithm, with 3000 iterations, and a perplexity of 20.0.

Article Snippet: For the tSNE analysis, FlowJo Downsample plugin was used to ensure similar number of events in all samples.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Comparative analysis of human gut- and blood-derived mononuclear cells: contrasts in function and phenotype

doi: 10.3389/fimmu.2024.1336480

Figure Lengend Snippet: FItSNE projection of the main cell groups among the various cytokine-expressing cell subtypes in PBMC and RMC. Layers of different cell populations were removed from the original tSNE projection for better visualization of the main cell groups in PBMC (n=8) and RMC (n=11); NK and other CD3- cells (A) , CD4 + and CD8 + T cells (B) , and γδ T cells are demonstrated (C) . FItSNE analysis was performed with the tSNE function in FlowJo, FItSNE algorithm, with 3000 iterations, and a perplexity of 20.0.

Article Snippet: For the tSNE analysis, FlowJo Downsample plugin was used to ensure similar number of events in all samples.

Techniques: Expressing

Journal: ImmunoHorizons

Article Title: Effect of a fructose-rich diet on gut microbiota and immunomodulation: potential factors for multiple sclerosis

doi: 10.4049/immunohorizons.2300008

Figure Lengend Snippet: Mice were fed an FRD or ND for 4 or 12 weeks before ileal lamina propria cells were isolated and analyzed by flow cytometry. tSNE analysis was performed on CD45+ cells previously gated on lymphocytes and single cells. (A) Schematic of the experimental procedure. (B) tSNE and FlowSOM analysis of flow cytometry data. (C) Table of selected markers expressed by the indicated populations taken from the tSNE analysis. (D) The number of cells in the indicated populations in each diet group. (E) Two-dimensional plot of Helios and RORγT in populations 3 and 4 from mice fed an FRD. N=5 per group, 2-way ANOVA for bar graphs.

Article Snippet: All flow cytometry data was collected using a Cytek Aurora (Cytek, Fremont, CA) and analyzed using the FlowJo tSNE package (FlowJo, Ashland, OR) with downsampling of CD45+ cells prior to tSNE analysis to normalize populations between samples (27).

Techniques: Isolation, Flow Cytometry

Journal: ImmunoHorizons

Article Title: Effect of a fructose-rich diet on gut microbiota and immunomodulation: potential factors for multiple sclerosis

doi: 10.4049/immunohorizons.2300008

Figure Lengend Snippet: Mice were fed an FRD or ND for 4 or 12 weeks before colonic lamina propria cells were isolated and analyzed by flow cytometry. tSNE analysis was performed on CD45+ cells previously gated on lymphocytes and single cells. (A) Schematic of the experimental procedure. (B) tSNE and FlowSOM analysis of flow cytometry data and (C) the number of cells in the indicated populations in each diet group. (D) Histograms from flow cytometry data depicting expression of select identifying markers by the selected populations. N=5 per group, 2-way ANOVA for bar graphs.

Article Snippet: All flow cytometry data was collected using a Cytek Aurora (Cytek, Fremont, CA) and analyzed using the FlowJo tSNE package (FlowJo, Ashland, OR) with downsampling of CD45+ cells prior to tSNE analysis to normalize populations between samples (27).

Techniques: Isolation, Flow Cytometry, Expressing

Journal: ImmunoHorizons

Article Title: Effect of a fructose-rich diet on gut microbiota and immunomodulation: potential factors for multiple sclerosis

doi: 10.4049/immunohorizons.2300008

Figure Lengend Snippet: 4-6 week old mice were fed an FRD for 4 or 12 weeks before splenic immune cells were isolated and analyzed by flow cytometry. (A) Schematic of the experiment. tSNE analysis was performed on CD45+ cells previously gated on lymphocytes and single cells. (B) tSNE and FlowSOM analysis of splenic immune populations in each diet group. (C) Number of cells in selected populations in each diet group. (D) Histograms from flow cytometry depicting prominent identifying markers of the selected populations. N=5 per group, 2-way ANOVA for bar graphs.

Article Snippet: All flow cytometry data was collected using a Cytek Aurora (Cytek, Fremont, CA) and analyzed using the FlowJo tSNE package (FlowJo, Ashland, OR) with downsampling of CD45+ cells prior to tSNE analysis to normalize populations between samples (27).

Techniques: Isolation, Flow Cytometry