145nd cd69 1 h1 2 f3  (fluidigm)

Journal: Cellular and Molecular Life Sciences: CMLS

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

doi: 10.1007/s00018-024-05423-9

Figure Lengend Snippet: Lamin A/C deficiency in matured GM-CSF-BMDCs reduces CD4 T cell activation, proliferation and Th1 differentiation in vitro and in vivo. 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 OVA 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). This schematic was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution CC BY 4.0 License; https://smart.servier.com

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, PE-Cy7; 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, -VB711; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin, -V450; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Activation Assay, In Vitro, In Vivo, Flow Cytometry, Derivative Assay, Sonication

Journal: bioRxiv

Article Title: Glutathione supersulphide regulates T-cell receptor signalling

doi: 10.1101/2024.04.30.591985

Figure Lengend Snippet: a-d, Expression of surface antigens CD25 and CD69 on naïve CD4 + T cells obtained from WT and Cars2 +/– mice as analysed by flow cytometry (FCM) at 24 h after stimulation with anti-CD3ε antibody, or without stimulation, in the presence or absence of anti-CD28 antibody. Representative FCM plots and MFI (mean fluorescence intensity) of surface expression of CD25 on CD4 + T cells ( a and b ), and representative FCM plots and surface expression of CD69 on CD4 + T cells ( c and d ). e, IL-2 released into the culture medium by activated CD4 + T cells obtained from WT and Cars2 +/– mice analysed via ELISA. f-i, FCM analysis of phosphorylation of TCR/CD3 complex components, including CD3ζ, ZAP-70, and ERK, in splenocytes obtained from WT and Cars2 +/– mice after stimulation with anti-CD3ε antibody. Splenocytes were stimulated with an anti-CD3ε antibody, or were not stimulated, and were then cross-linked with goat anti-Armenian hamster IgG for 1 min. Representative FCM plots of phosphorylation of CD3ζ, ZAP-70, and ERK in CD4 + T cells ( f ), and MFIs of pCD3ζ ( g ), pZAP-70 ( h ), and pERK ( i ) relative to that in unstimulated WT cells. Data are mean ± SD. P values were analysed with one-way ANOVA with Tukey’s test. * P < 0.05; ** P < 0.01, *** P < 0.001. Data represent at least two independent experiments.

Article Snippet: Anti-CD4 (RM4-5) and anti-CD69 (H1.2F3) antibodies were purchased from Tonbo Biosciences.

Techniques: Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Phospho-proteomics