Journal: Scientific Reports

Article Title: An Essential Role for the Transcription Factor Runx1 in T Cell Maturation

doi: 10.1038/srep23533

Figure Lengend Snippet: ( a ) DP and CD4 SP thymocytes from Rag1-GFP WT and Rag1-GFP CD4-cre Runx1 cKO mice were examined for surface expression of TCRβ, CD5 and intracellular expression of Bcl-xL, Bcl-2. ( b ) DP and CD4 SP thymocytes from Rag1-GFP WT and Rag1-GFP CD4-cre Runx1 cKO mice were examined for intranuclear expression of ThPOK, c-Rel, RORγ, and surface expression of CXCR4. ( c ) DP and CD4 SP thymocytes from Rag1-GFP WT and Rag1-GFP CD4-cre Runx1 cKO mice were examined for intranuclear expression of Nur77, Helios, and intracellular expression of Bim. For surface and intracellular FACS, thymocytes were first gated on Rag1-GFP + to exclude recirculating mature T cells from the analysis. For intranuclear FACS, as fluorescence of Rag1-GFP protein is disrupted by the use of Foxp3/Transcription factor buffer, thymocytes were not gated for Rag1-GFP expression. Grey histogram represents DP thymocytes while solid line represents CD4 SP thymocytes. Data shown are representative of at least four Rag1-GFP WT and five Rag1-GFP CD4-cre Runx1 cKO mice from four independent experiments.

Article Snippet: The following antibodies were purchased from BD Bioscience, BioLegend, eBioscience, R&D Systems, Tonbo Bioscience, Cell Signaling Technology or Cedarlane: Bcl-2 (BCL/10C4), Bcl-xL (7B2.5), Bim (H-191), CCR4 (2G12), CCR7 (4B12), CCR9 (9B1), CD4 (RM4-5, GK1.5), CD45.1 (A20), CD5 (53-7.3), CD8α (53-6.7), CD24 (M1/69), CD44 (IM7), CD45RB (C363-16A), CD55 (RIKO-3), CD62L (MEL-14), CD69 (H1.2F3), complement C1q (RMC7H8), complement C3 (RMC11H9), complement C4 (RMC16D2), CXCR4 (2B11), Helios (22F6), IgM (RMM-1), IL-7Rα (A7R34), Nur77 (12.14), c-Rel (1RELAH5), RORγ (B2D), recombinant mSiglec-E-Fc chimera, Runx1 (RXDMC), S1P1 (713412), TCRβ (H57-597), ThPOK (2POK), TNF-α (MP6-XT22) and Qa2 (695H1-9-9).

Techniques: Expressing, Fluorescence

Journal: Biomedicines

Article Title: Analysis of Primary Chronic Lymphocytic Leukemia Cells’ Signaling Pathways

doi: 10.3390/biomedicines12030524

Figure Lengend Snippet: ( A ) Analysis of BCL2 expression in a representative CLL blood sample (top) and in a peripheral blood sample from healthy control (bottom) using flow cytometry. The cell subpopulations according to the expression of CD5 and CD19 were analyzed for BCL2 expression. ( B ) Mean fluorescence intensity for each population calculated by normalizing the MFI of a gene with a corresponding isotype control. The bars represent the mean and SEM of 10 patients’ samples. The p -value is indicated in the figure. BCL2 expression (black line) and the according isotype control (gray line).

Article Snippet: Cells were washed in PBS (supplemented with 2% FCS), labeled with primary antibodies for 20 min, washed, and, when appropriate, labeled with a secondary anti-mouse IgG2b Alexa Fluor647 antibody (Invitrogen, Carlsbad, CA, USA) for 20 min. Antibodies to CD5 (TONBO), CD19 (TONBO and eBioscience), BCL-2 (BD), HES1 (Abnova), and NOTCH1 (LSBio) were used [ , ].

Techniques: Expressing, Control, Flow Cytometry, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: RRAS2 shapes the TCR repertoire by setting the threshold for negative selection

doi: 10.1084/jem.20181959

Figure Lengend Snippet: Enhanced expression of positive selection and negative selection markers in thymocytes from Rras2 − /− mice. (A) Thymocytes from AND TCR transgenic mice in mixed k/b background were analyzed for expression of positive selection markers CD5 and CD69 within the DP and CD4SP subsets. Bar plots show quantitative data from a total of three experiments. Percentage and mean fluorescence intensity (MFI) values are represented normalized to those of the mean values in the WT controls within each experiment. Mean percentage and MFI in WT thymocytes are set to 1. Quantitative data are means ± SEM ( n = 7–12 mice per group). *, P < 0.05; **, P < 0.005 (unpaired two-tailed Student’s t test). This experiment was repeated three times. (B) Expression of positive selection markers CD5 and CD69 in the thymic subsets of CD45.2 + thymocytes from the bone marrow competition experiments of . An example of the gating strategy is shown for the DP population. Quantitative MFI data for the entire DP, CD4SP, and CD8SP populations are means ± SEM ( n = 5 mice per group). *, P < 0.05 (unpaired two-tailed Student’s t test). This experiment was repeated twice. (C) Thymocytes from AND TCR transgenic mice in mixed k/b background were analyzed for expression of the negative selection marker Nur77 within CD4 + subsets, as indicated. Bar plots show the normalized expression of Nur77 in the CD8 high (DP) and CD8 low (transitional and CD4SP) subpopulations in a combination of three experiments. MFI values are represented normalized to those of the mean values in the WT controls within each experiment. Quantitative data are means ± SEM ( n = 8–9 mice per group). **, P < 0.005 (unpaired two-tailed Student’s t test). This experiment was repeated three times. (D) RT-qPCR analysis of gene expression in sorted DP thymocytes from Rras2 +/+ and Rras2 − /− mice. Genes are grouped according to their differential expression during positive and negative selection . An interleaved box and whiskers plot showing all data points and the maximum and minimum has been chosen for data representation. Values are normalized to Rras2 +/+ . *, P < 0.05. An unpaired two-tailed Student’s t test was used to compare significance of individual genes; a two-way ANOVA test was used to compare gene sets. This experiment was repeated four times. n.s., not significant.

Article Snippet: The following antibodies were used: anti-mouse CD45R-V450 -biotin -APC (RA3-6B2), CD4-PerCP -647 -FITC (RM4-5), CD8-biotin -V405 -PerCP -647 (53–6.7), CD11b-biotin (M1/70), purified CD16/32 (2,4G2), CD19–PE-Cy7 (1D3), CD25-APC (3C7), CD43-biotin (S7), CD45.1–APC-Cy7 (A20), CD45.2-APC (104), Gr1-biotin (RB6-8C5), CD44-PE (IM7), TCRβ-FITC (H57-597), CD3ε-PerCP (2C11), Vβ3-biotin -PE (KJ25), CD5-PE -biotin (53–7.3), IL-17a–PerCP (TC11-18H10), CD69-FITC -PerCP (H1.2F3), and NK1.1-biotin from BD Pharmigen; anti-mouse F4/80–biotin (BM8), H2-Kk–V405 (AF3-12.1.3), Foxp3-PE (NRRF-30), and Nur77-PE (12.14) from eBioscience; anti-mouse IFNγ–APC from Milteny; anti-mouse Vα2–PerCP (B20.1) from BioLegend; rabbit anti-mouse pERK (T202 Y204), pAKT (S473), pS6 (S240), total ERK, and total CD3ε from Cell Signaling.

Techniques: Expressing, Selection, Transgenic Assay, Fluorescence, Two Tailed Test, Marker, Quantitative RT-PCR