Journal: bioRxiv

Article Title: The CNS lymphatic system modulates the adaptive neuro-immune response in the perilesional cortex in a mouse model of traumatic brain injury

doi: 10.1101/821645

Figure Lengend Snippet: Pseudocolor dot plots (A) and (B) represent gated subpopulations CD69 vs. CD44 of CD4+ and CD8+, respectively. Stacked bargrams in (C) and (D) show respectively the counts and frequencies of CD8+ T cell subpopulations, as analyzed in the perilesional cortices of WT and TG mice. No significant differences in CD8+ subpopulations were found between genotypes. In CD4+ subpopulation, instead, we observed a significant reduction in the counts of CD44 hi CD69+ and CD44 hi CD69-subpopulations (E) , in K14-VEGFR3-Ig compared to WT mice. However, no differences were observed in the different subpopulation frequencies (F) . Data are presented as mean ± s.e.m. A binomial negative regression was applied to assess statistical differences in the counts of total T cells between WT ipsi and TG ipsi. The Kruskal Wallis test was used for the analysis of frequency distribution. #p < 0.05; *p < 0.05 vs. WT ipsi.

Article Snippet: Antibodies used: TCRβ PE-Cy7 (1:100 or 1:200 clone H57-597), CD44 PE (1:300 clone IM7) (both BioLegend); CD8a APC-R700 (1:150 or 1:200, clone 53-6.7), CD69 BV421 (1:100, clone H1.2F3), CD25 BB515 (1:150, clone PC61) (BD Biosciences); CD4 FITC (1:500, clone RM4-5), CD4 eFluor506 (1:500, clone RM4-5), CD8 PerCP eFluor710 (1:300, clone 53-6.7), CD44 APC (1:300 or 1:400, clone IM7), FoxP3 (1:40, clone FJK-16s) (eBioscience Thermo Fisher Scientific, Waltham, MA, USA); CD69 APC (1:20, clone H1.2F3, Miltenyi Biotech).

Techniques:

Journal: PLoS ONE

Article Title: A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

doi: 10.1371/journal.pone.0000353

Figure Lengend Snippet: (A) Lymph nodes cells from 6–8 week old Ag pos and Ag neg mice were stained with antibodies against CD4, CD8 and Vβ7 and analysed by flow cytometry. Numbers in quadrants are percentage of live-gated lymphocytes. (B) Lymph node cells were stained with antibodies against CD8, Vβ7 and the activation markers CD44, CD62L, CD69 and CD25. Histograms display gated viable CD8 + Vβ7 + cells. Numbers in histograms represent the specific MFI of the activation marker staining for the gated population. Data are representative of at least three independent experiments.

Article Snippet: The following mAbs were used for flow cytometric staining: rat-anti-mouse Vβ7 FITC (Serotec, UK), rat-anti-mouse CD4 FITC, rat-anti-mouse CD8α APC, rat-anti-mouse CD8α Cy-Chrome 5 (CY-5), rat-anti-mouse Vβ7 PE, hamster-anti-mouse CD69 PE, rat-anti-mouse CD25 PE, rat-anti-mouse CD62L-FITC, rat-anti-mouse CD44 PE, rat-anti-mouse CD43 activation-associated glycoform-PE (all BD Biosciences).

Techniques: Staining, Flow Cytometry, Activation Assay, Marker

Journal: bioRxiv

Article Title: CCR5 deficiency impairs CD4 + T cell memory responses and antigenic sensitivity through increased ceramide synthesis

doi: 10.1101/2020.02.14.948893

Figure Lengend Snippet: A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition ( n = 3). B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B . Data shown as mean ± SEM of at least 4 independent experiments. D. Cer levels in OT-II 10-day lymphoblasts, untreated or treated with SMase ( n = 2). E. Representative small field image showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles, and statistical analysis. F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS. G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F . Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells. Data shown as mean ± SEM ( n = 3). H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G . Filters were rehybridized with β-actin as loading control. I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (black; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size, and statistical analysis. J. Percentage of CD69 + shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779. Data shown as mean ± SEM. * p <0.05, ** p <001, one-tailed ( I ) or two-tailed unpaired Student’s t -test. Bar, 50 nm.

Article Snippet: Antibodies used for characterization of mouse cells by flow cytometry were anti-Vα 2 TCR-PE (B20.1), -CD25-PE (PC61), -CD45.2-FITC (104), -CD62L-FITC/APC (MEL-14), -CD69-PeCy7 (H1.2F3) and biotinylated anti-CXCR5 (2G8) from BD-Biosciences; anti human biotinylated CD3 (OKT3), anti-CD4-PeCy7/efluor450/PacificBlue (RM4.5), -IFNγ-APC (XMG1.2), and -PD1-efluor780 (J43) from eBioscience, and anti-CD44-PeCy5/APC (IM7) from BioLegend.

Techniques: Generated, Western Blot, Marker, Lysis, Labeling, Expressing, Selection, One-tailed Test, Two Tailed Test

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hamster monoclonal anti-mouse CD69 - 143Nd , Fluidigm , Cat#3143004B; RRID:AB_2827881.

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Reverse Transcription, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ+ cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna -/- -BMDCs was undertaken with (A) sonicated VACV extract, (B) Pam3CSK4, or (C) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA323–339 cognate OT-IIp. (D) WT- or Lmna -/- -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 (A and B, n=3) or out of 4 (C, n=4) or out of 2 (D, n=4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Derivative Assay, Sonication, Activation Assay