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

Journal: Viruses

Article Title: Characterization of Natural Killer Cell Profile in a Cohort of Infected Pregnant Women and Their Babies and Its Relation to CMV Transmission

doi: 10.3390/v16050780

Figure Lengend Snippet: Comparative analysis of NK cell profile in CMV-transmitting vs. non-transmitting mothers. ( A,B ) Scatter plots with bar (mean ± SD) depict frequency distribution, as in B,C. Unpaired t test and Mann–Whitney test were used to assess differences in cell frequencies between transmitting (red dots, n = 8) and non-transmitting (blue dots, n = 9) mothers. Significance was set at p < 0.05. ( C ) tSNE algorithm was configured to distribute data combined from transmitting and non-transmitting mothers’ samples according to the expression of NK cell markers CD56, CD16, NKG2C, NKG2A, CD57, NKG2D, DNAM-1, KIRs, and PD-1. Multigraph histogram overlays with geomean values were generated in a combined FCS file obtained by concatenating 2000 events in NK cell down-sample (identified as CD3-CD19-CD14- live lymphocytes) of transmitting (red, n = 7) and non-transmitting (blue, n = 8) mothers. ( D ) By using the same combined FCS file, single parameter heatmaps were obtained for transmitting (upper panels) and non-transmitting (lower panels) groups. Outline population (black circle) is specific to non-transmitting mothers and only the expressed markers are reported. TD NK: terminally differentiated NK; ML NK: memory-like NK.

Article Snippet: The “DownSample” FlowJo plugin was run on NK cells in order to reduce and make uniform the population sizes for the further concatenation of samples from different groups into a single FCS file. tSNE was performed on the concatenated file using the “TSNE” plugin and the maps generated using data from the following compensated parameters as inputs: CD56, CD16, NKG2C, NKG2A, NKG2D, DNAM-1, CD57, KIR2DL1/S1/S3/S5, KIR2DL2/L3, and PD-1 for the phenotype analysis and CD56, CD16, NKG2C, DNAM-1, CD57, NKp46, PD-1, and CD107a for the degranulation analysis; under the following tSNE settings: iteration 1000, perplexity 30, learning rate (Eta) 910–2100, the ANNOY algorithm as the k nearest neighbors (KNN) algorithm and the fast Fourier transform (FTT) interpolation as the gradient algorithm, resulting in tSNE plots with less than 5 million events ( ).

Techniques: MANN-WHITNEY, Expressing, Generated

Journal: Viruses

Article Title: Characterization of Natural Killer Cell Profile in a Cohort of Infected Pregnant Women and Their Babies and Its Relation to CMV Transmission

doi: 10.3390/v16050780

Figure Lengend Snippet: Comparative analysis of NK cell profile in newborns with or without CMV congenital infection. ( A , B ) Scatter plots with bar (mean ± SD) depict frequency distribution, as in B,C. Mann–Whitney test was used to assess differences in cell frequencies between congenital (purple dots, n = 12) and non-congenital (pink dots, n = 5) newborns. Significance was set at p < 0.05. ( C ) tSNE algorithm was configured to distribute data combined from congenital and non-congenital children’s samples according to C. Multigraph histogram overlays with geomean values were generated in a combined FCS file obtained by concatenating 1880 events in NK cell down-sample of congenital (purple, n = 11) and non-congenital (pink, n = 5) children. ( D ) By using the same combined FCS file, single parameter heatmaps were obtained for congenital (upper panels) and non-congenital (lower panels) groups. Outline population (black circle) is specific to congenital children and only the expressed markers are reported. TD NK: terminally differentiated NK; ML NK: memory-like NK.

Article Snippet: The “DownSample” FlowJo plugin was run on NK cells in order to reduce and make uniform the population sizes for the further concatenation of samples from different groups into a single FCS file. tSNE was performed on the concatenated file using the “TSNE” plugin and the maps generated using data from the following compensated parameters as inputs: CD56, CD16, NKG2C, NKG2A, NKG2D, DNAM-1, CD57, KIR2DL1/S1/S3/S5, KIR2DL2/L3, and PD-1 for the phenotype analysis and CD56, CD16, NKG2C, DNAM-1, CD57, NKp46, PD-1, and CD107a for the degranulation analysis; under the following tSNE settings: iteration 1000, perplexity 30, learning rate (Eta) 910–2100, the ANNOY algorithm as the k nearest neighbors (KNN) algorithm and the fast Fourier transform (FTT) interpolation as the gradient algorithm, resulting in tSNE plots with less than 5 million events ( ).

Techniques: Infection, MANN-WHITNEY, Generated

Journal: Viruses

Article Title: Characterization of Natural Killer Cell Profile in a Cohort of Infected Pregnant Women and Their Babies and Its Relation to CMV Transmission

doi: 10.3390/v16050780

Figure Lengend Snippet: NK cell degranulation ( A ) tSNE algorithm was generated for CD107a+ NK cells and configured to distribute data combined from study cohorts of stimulated samples according to the expression of NK cell markers CD56, CD16, CD107a, NKG2C, CD57, NKp46, DNAM-1, and PD-1. Multigraph histogram overlays with geomean values were generated in two combined FCS files. One was obtained by concatenating 1000 events in NK cell down-sample of either CMV-infected (orange, n = 9) and uninfected (green, n = 4) pregnant women or transmitting (red, n = 5) and non-transmitting (blue, n = 4) mothers. The other one was obtained by concatenating 1180 events in the NK cell down-sample of congenital (purple, n = 7) and non-congenital (pink, n = 5) children. ( B ) By using the same combined FCS files, single parameter heatmaps were obtained for each study group. Outline populations (black circles) are specific to infected pregnant women and congenital children, and only the expressed markers are reported.

Article Snippet: The “DownSample” FlowJo plugin was run on NK cells in order to reduce and make uniform the population sizes for the further concatenation of samples from different groups into a single FCS file. tSNE was performed on the concatenated file using the “TSNE” plugin and the maps generated using data from the following compensated parameters as inputs: CD56, CD16, NKG2C, NKG2A, NKG2D, DNAM-1, CD57, KIR2DL1/S1/S3/S5, KIR2DL2/L3, and PD-1 for the phenotype analysis and CD56, CD16, NKG2C, DNAM-1, CD57, NKp46, PD-1, and CD107a for the degranulation analysis; under the following tSNE settings: iteration 1000, perplexity 30, learning rate (Eta) 910–2100, the ANNOY algorithm as the k nearest neighbors (KNN) algorithm and the fast Fourier transform (FTT) interpolation as the gradient algorithm, resulting in tSNE plots with less than 5 million events ( ).

Techniques: Generated, Expressing, Infection

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