Journal: PLoS Pathogens

Article Title: Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection

doi: 10.1371/journal.ppat.1007611

Figure Lengend Snippet: Ileum and colon tissues were collected from mouse recipients at 21 days post gavage and analyzed for frequencies of CD69+ and CD103+ T cells. (A) Gating strategy for CD69+ T cells. Representative plots from ileum tissues are shown. (B-C) Frequencies of (B) CD69+ CD8+ T cells, (C) CD103+ CD8+ T cells, and (D) CD103+ CD4+ T cells in the ileum. (E) Frequencies of CD69+ CD4+ T cells in the colon. Each data point represents a single mouse gavaged with an individual donor’s feces, and a representative mouse was used for stools tested in replicate mice. Lines represent medians. Statistical analyses were performed using t-tests to compare groups if data from both groups had normal distributions and Mann-Whitney tests to compare groups if data from at least one group had a non-parametric distribution. ** = p<0.01, * = p<0.05.

Article Snippet: 0.5–1 x 10 6 ileum and colon cells from each mouse were stained in FACS buffer for 30 min at 4° C with the following antibodies: CD3-PerCP-Cy5.5 (17A2, Biolegend), CD4-FITC (RM4-4, eBioscience), CD8-BV510 (53–6.7, Biolegend), CD69-PE-Cy7 (h1.2F3, eBioscience), CD103-PE-Dazzle-594 (2E7, Biolegend).

Techniques: MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection

doi: 10.1371/journal.ppat.1007611

Figure Lengend Snippet: mLN from mouse recipients were collected and analyzed for effector memory CD4+ T cell frequency (CD44+ CD62L-) using flow cytometry. (A) Comparison of frequencies of CD44+ CD62- CD4+ T cells in the mLN across recipient groups. (B) Spearman correlations of effector memory CD4+ T cell (T em ) frequencies in the mLN with frequencies of CD69+ CD4+ T cells in the colon. Each data point represents a single mouse gavaged with an individual donor’s feces, and a representative mouse was used for stools tested in replicate mice. Lines represent medians. Statistical analyses were performed using t-tests to compare groups if data from both groups had normal distributions and Mann-Whitney tests to compare groups if data from at least one group had a non-parametric distribution. * = p<0.05.

Article Snippet: 0.5–1 x 10 6 ileum and colon cells from each mouse were stained in FACS buffer for 30 min at 4° C with the following antibodies: CD3-PerCP-Cy5.5 (17A2, Biolegend), CD4-FITC (RM4-4, eBioscience), CD8-BV510 (53–6.7, Biolegend), CD69-PE-Cy7 (h1.2F3, eBioscience), CD103-PE-Dazzle-594 (2E7, Biolegend).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection

doi: 10.1371/journal.ppat.1007611

Figure Lengend Snippet: Microbial correlates with donor/recipient T cell activation and gut homing, and in vitro HIV infection.

Article Snippet: 0.5–1 x 10 6 ileum and colon cells from each mouse were stained in FACS buffer for 30 min at 4° C with the following antibodies: CD3-PerCP-Cy5.5 (17A2, Biolegend), CD4-FITC (RM4-4, eBioscience), CD8-BV510 (53–6.7, Biolegend), CD69-PE-Cy7 (h1.2F3, eBioscience), CD103-PE-Dazzle-594 (2E7, Biolegend).

Techniques: Activation Assay, In Vitro, Infection

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Reagents Used for This Study

Article Snippet: Anti‐CD69 PE‐Cy7 , H1.2F3 , 1:200, 1:1000 , 10 , ThermoFisher , 25‐0691‐82.

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

doi: 10.1083/jcb.201305088

Figure Lengend Snippet: SKAP55 is required for microcluster persistence and movement, and for contact stability. (A) Confirmation of the efficacy of SKAP55 knockdown and add-back in stable SKAP55 knockdown cells (JSKAP.SY) with or without transient reconstitution ( n = 3). (B) J14.SY and JSKAP.SY cells expressing mRFP1 or SKAP55.mRFP1 were stimulated on coverslips and imaged for at least 5 min. Representative MOT images and kymographs are shown. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. See for microcluster properties and experiment numbers. (C) Composite microcluster traces for the conditions examined in B. Numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (D) Fraction of the contact area engaged in fluctuation (see Fig. S1 , F and G; n = 3). (E) Fraction of cells scored as displaying unstable contacts (see Fig. S1, I and J; n = 5). (F) Surface expression of CD69 in J14.SY and JSKAP.SY cells, after normalization to a TCR-stimulated J14.SY control ( n = 4). (G) Kinetics of Erk1/2 phosphorylation in J14.SY and JSKAP.SY stimulated for the indicated time points ( n = 3). Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): **, P < 0.01. From JSKAP.SY (with or without mRFP1): ##, P < 0.01.

Article Snippet: Western blotting was conducted with antibodies directed against ADAP (BD), β1 integrin (EP1041Y; Abcam), CD69-PE/Cy7 (No. 310912; BioLegend), Phospho Erk1/2 (No. 4370; Cell Signaling Technology), total Erk 1/2 (No. 4695; Cell Signaling Technology), Flag (M2; Sigma-Aldrich), RFP (ACT-CM-MRRFP10; Allele), GFP (JL-8; Takara Bio Inc.), γ-tubulin (Sigma-Aldrich), Mst1 (Epitomics), RapL (Sigma-Aldrich), RIAM (Epitomics), SKAP55 (gift from B. Schraven [University of Magdeburg, Magdeburg, Germany] and S. Kliche [Otto von Guerike University, Magdeburg, Germany]; targets SKAP55 DM), SKAP55 (targets SKAP55 PH-SH3 linker; BD), and talin (8D4; Sigma-Aldrich).

Techniques: Knockdown, Expressing, Control, Phospho-proteomics

Journal: Biology of Reproduction

Article Title: Immune response to a model shared placenta/tumor-associated antigen reduces cancer risk in parous mice †

doi: 10.1095/biolreprod.116.144907

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: Cells were detected by flow cytometry using Streptavidin-APC (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Fluorophore Vendor; catalog number CD8 Pacific blue Biolegend; 100725 CD19 APC Biolegend; 115512 CD44 APC-Cy7 Biolegend; 103028 ICOS PE-Cy5 Biolegend; 107708 CD69 PE-Cy7 eBioscience; 28-0691-82 PD-1 FITC eBioscience; 11-9981-82 OVA-H2K b Streptavidin-APC J. Yewdell, NIH, clone 25-D1.16 Rat IgG2aκ Pacific Blue Biolegend; 400527 Rat IgG2aκ APC Pharmingen; 553932 Rat IgG2bκ APC-Cy7 Biolegend; 400623 Syrian hamster IgG PE-Cy5 Biolegend; 402009 Armenian hamster IgG PE-Cy7 eBioscience; 25-4888-82 Rat IgG2a FITC BD Biosciences; 553929 Open in a separate window Antibodies used for flow cytometry.

Techniques: Cytometry

Journal: Clinical & Translational Immunology

Article Title: Tissue‐specific tumor microenvironments influence responses to immunotherapies

doi: 10.1002/cti2.1094

Figure Lengend Snippet: Mammary fat pad (MFP) and lung tumors have distinct TMEs that are altered differentially by αPD‐1/αCTLA4 treatment. BALB/c mice bearing MFP or lung tumors (4 × 10 5 67NR cells in MFP or IV) were treated with αPD‐1/αCTLA4 and harvested 7 days post‐treatment initiation. (a) Flow cytometry of immune cell populations indicated as a percentage of live CD45.2 + cells within MFP or lung tumors treated with isotype antibody (NT) or αPD‐1/αCTLA4 (T), or organs without tumor (naïve). Pooled data from 2 independent experiments, n = 8–11/group. Mann–Whitney U‐ test. (b) Heatmap of gene expression in MFP and lung tumors of genes in KEGG NK cell‐mediated cytotoxicity pathway. RNA was extracted from tumors harvested from BALB/c mice injected with 4 × 10 5 67NR cells in the MFP or IV (lung mice), 14 days after injection. n = 5 tumors/group. (c) Expression of CD69 on live CD45.2 + CD3 − DX5 + cells by flow cytometry. Mann–Whitney U‐ test. (d) Maturation of NK cells (live CD45.2 + CD3 − DX5 + ) assessed by expression of CD11b and CD27 by flow cytometry. (e) Expression of IFNγ by NK cells and CD8 + T cells stimulated ex vivo by flow cytometry. ns P ≥ 0.05 * P < p0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: The following antibodies were used: CD45.2 APC Cy7 (eBioscience 104), CD3 BV605 (Biolegend 17A2), CD8 BUV737 (BD 53‐6.7), CD8 BV711 (Biolegend, 53‐6.7), CD4 BUV805 (BD GK1.5), CD4 BV785 (Biolegend GK1.5), FoxP3 e450 (eBioscience FJK16S), PD‐1 BUV395 (BD J43), 4‐1BB PE (BD 1AH2), CD69 PE Cy7 (eBioscience H1.2F3), CD19 BV785 (BD 6D5), CD49b FITC (BD DX5), CD11b BV711 (Biolegend M1/70), Ly6G BV605 (Biolegend, 1A8), Ly6C PE Cy7 (Biolegend HK1.4), F4/80 BV421 (BD T45‐2342), MHCII APC (eBioscience M5/114.1.5.2), CD11c BV785 (Biolegend N418), PD‐L1 BUV395 (BD M1H5), CD80 Biotin (BD 16‐10A1), CD86 Biotin (eBioscience GL1), DR5 Biotin (eBioscience MD5‐1), MHCI Biotin (BD H2K d ), IFNγ APC (Biolegend XMG1.2), CTLA4 APC (BD UC10‐4F10‐11), CD40 PE (BD 3/23) and Streptavidin BUV805 (BD) or e450 (eBioscience).

Techniques: Flow Cytometry, MANN-WHITNEY, Expressing, Injection, Ex Vivo