anti cd5  (Thermo Fisher)


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    Thermo Fisher anti cd5
    Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show <t>CD5</t> versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.
    Anti Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti cd5 - by Bioz Stars, 2023-02
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    1) Product Images from "A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage"

    Article Title: A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage

    Journal: eLife

    doi: 10.7554/eLife.80277

    Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show CD5 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.
    Figure Legend Snippet: Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show CD5 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.

    Techniques Used: Staining, Expressing, Two Tailed Test, MANN-WHITNEY, Selection


    Figure Legend Snippet:

    Techniques Used: Staining, Purification, Binding Assay, Magnetic Beads, Microscopy, Software

    anti cd5  (Thermo Fisher)


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    Thermo Fisher anti cd5
    Anti Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti cd5 - by Bioz Stars, 2023-02
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    cd5  (Thermo Fisher)


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    Thermo Fisher cd5
    Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd5 pb  (Thermo Fisher)


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    Thermo Fisher cd5 pb
    a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, <t>CD5,</t> CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).
    Cd5 Pb, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Cul5 E3 Ligase Complex Is a Key Negative Feedback Regulator of TCR/IL2 Signaling and Anti-Tumor Activity in CD8 + T Cells"

    Article Title: The Cul5 E3 Ligase Complex Is a Key Negative Feedback Regulator of TCR/IL2 Signaling and Anti-Tumor Activity in CD8 + T Cells

    Journal: bioRxiv

    doi: 10.1101/2022.11.16.516824

    a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, CD5, CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).
    Figure Legend Snippet: a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, CD5, CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).

    Techniques Used: Mass Spectrometry, Flow Cytometry, Expressing, Activation Assay, Western Blot, Irradiation, Adoptive Transfer Assay

    anti dog cd5 apc  (Thermo Fisher)


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    Thermo Fisher anti dog cd5 apc
    Anti Dog Cd5 Apc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd5  (Thermo Fisher)


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    Thermo Fisher cd5
    Antibodies for flow cytometry (FC) analysis used in the case.
    Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mott Cell Differentiation in Canine Multicentric B Cell Lymphoma with Cross-Lineage Rearrangement and Lineage Infidelity in a Dog"

    Article Title: Mott Cell Differentiation in Canine Multicentric B Cell Lymphoma with Cross-Lineage Rearrangement and Lineage Infidelity in a Dog

    Journal: Veterinary Sciences

    doi: 10.3390/vetsci9100549

    Antibodies for flow cytometry (FC) analysis used in the case.
    Figure Legend Snippet: Antibodies for flow cytometry (FC) analysis used in the case.

    Techniques Used: Flow Cytometry

    cd5 percpcy5 5  (Thermo Fisher)


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    Thermo Fisher cd5 percpcy5 5
    Cd5 Percpcy5 5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd5  (Thermo Fisher)


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    Thermo Fisher cd5
    Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd5  (Thermo Fisher)


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    Thermo Fisher cd5
    Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd5/product/Thermo Fisher
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    anti cd5  (Thermo Fisher)


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    Thermo Fisher anti cd5
    a Proportions of CD11a hi CD44 + T cells among CD8 - TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 5 for uninfected; n = 10 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of 2 independent experiments. ns not significant, p > 0.9999, ** p = 0.0047, 0.0049 (Kruskal–Wallis test and Dunn’s multiple comparison test). b Proportions of CD11a hi CD49d + T cells among CD8 - TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 5 for uninfected; n = 10 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of two independent experiments. ns not significant, p = 0.5205 ** p = 0.0024, * p = 0.0376 (Kruskal–Wallis test and Dunn’s multiple comparisons test). c <t>CD5</t> MFI on CD11a hi CD44 + T cells among CD8 − TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 11 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM. and are a summary of two independent experiments **** p < 0.0001 (two-tailed Mann–Whitney U -test). d Nur77 MFI on CD11a hi CD44 + T cells among CD8 − TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 7 for Cd4 +/+ ; n = 10 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of two independent experiments, *** p = 0.0007 (two-tailed Mann–Whitney U -test). e Representative FACS contour plot showing Tbet expression among CD4 + CD8 - TCRβ + T cells from the popliteal dLNs of mice, 28 days p.i. Data shown are representative of >3 independent experiments. f Proportion and numbers of Tbet+ cells among CD8 - TCRβ + CD11a hi CD44 + T cells from the popliteal dLNs of mice, 28 days p.i. ( n = 7 for Cd4 +/+ ; n = 10 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are a summary of two independent experiments. *** p = 0.0001 (two-tailed Mann–Whitney U-test). g Numbers of Tbet+ cells among CD8 - TCRβ + CD11a hi CD44 + T cells from the popliteal dLNs of mice, 28 days p.i. ( n = 6/group) ** p = 0.0087 (two-tailed Mann–Whitney U-test). h Representative FACS contour plot showing GP 66–77 tetramer+ or CLIP-tetramer+ CD4 T cells (control) among splenocytes of LCMV-infected animals 8d p.i. i Quantification of data shown in h ( n = 5 for CLIP Tet + ; n = 10 for Cd4 +/+ ; n = 8 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data are representative of two independent experiments. ns not significant, p = 0.8098 (two-tailed Mann–Whitney U-test). j Gating strategy to detect GP 66–77 Tetramer+ cells among TCRβ + CD8- CD4 + and TCRβ + CD8- CD4- T cells. k Quantification of data shown in j ( n = 8). Data are representative of two independent experiments. *** p < 0.001 (two-tailed Mann–Whitney U-test).
    Anti Cd5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd5/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    1) Product Images from "CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element"

    Article Title: CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element

    Journal: Nature Communications

    doi: 10.1038/s41467-022-28914-4

    a Proportions of CD11a hi CD44 + T cells among CD8 - TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 5 for uninfected; n = 10 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of 2 independent experiments. ns not significant, p > 0.9999, ** p = 0.0047, 0.0049 (Kruskal–Wallis test and Dunn’s multiple comparison test). b Proportions of CD11a hi CD49d + T cells among CD8 - TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 5 for uninfected; n = 10 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of two independent experiments. ns not significant, p = 0.5205 ** p = 0.0024, * p = 0.0376 (Kruskal–Wallis test and Dunn’s multiple comparisons test). c CD5 MFI on CD11a hi CD44 + T cells among CD8 − TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 11 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM. and are a summary of two independent experiments **** p < 0.0001 (two-tailed Mann–Whitney U -test). d Nur77 MFI on CD11a hi CD44 + T cells among CD8 − TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 7 for Cd4 +/+ ; n = 10 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of two independent experiments, *** p = 0.0007 (two-tailed Mann–Whitney U -test). e Representative FACS contour plot showing Tbet expression among CD4 + CD8 - TCRβ + T cells from the popliteal dLNs of mice, 28 days p.i. Data shown are representative of >3 independent experiments. f Proportion and numbers of Tbet+ cells among CD8 - TCRβ + CD11a hi CD44 + T cells from the popliteal dLNs of mice, 28 days p.i. ( n = 7 for Cd4 +/+ ; n = 10 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are a summary of two independent experiments. *** p = 0.0001 (two-tailed Mann–Whitney U-test). g Numbers of Tbet+ cells among CD8 - TCRβ + CD11a hi CD44 + T cells from the popliteal dLNs of mice, 28 days p.i. ( n = 6/group) ** p = 0.0087 (two-tailed Mann–Whitney U-test). h Representative FACS contour plot showing GP 66–77 tetramer+ or CLIP-tetramer+ CD4 T cells (control) among splenocytes of LCMV-infected animals 8d p.i. i Quantification of data shown in h ( n = 5 for CLIP Tet + ; n = 10 for Cd4 +/+ ; n = 8 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data are representative of two independent experiments. ns not significant, p = 0.8098 (two-tailed Mann–Whitney U-test). j Gating strategy to detect GP 66–77 Tetramer+ cells among TCRβ + CD8- CD4 + and TCRβ + CD8- CD4- T cells. k Quantification of data shown in j ( n = 8). Data are representative of two independent experiments. *** p < 0.001 (two-tailed Mann–Whitney U-test).
    Figure Legend Snippet: a Proportions of CD11a hi CD44 + T cells among CD8 - TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 5 for uninfected; n = 10 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of 2 independent experiments. ns not significant, p > 0.9999, ** p = 0.0047, 0.0049 (Kruskal–Wallis test and Dunn’s multiple comparison test). b Proportions of CD11a hi CD49d + T cells among CD8 - TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 5 for uninfected; n = 10 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of two independent experiments. ns not significant, p = 0.5205 ** p = 0.0024, * p = 0.0376 (Kruskal–Wallis test and Dunn’s multiple comparisons test). c CD5 MFI on CD11a hi CD44 + T cells among CD8 − TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 11 for Cd4 +/+ ; n = 12 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM. and are a summary of two independent experiments **** p < 0.0001 (two-tailed Mann–Whitney U -test). d Nur77 MFI on CD11a hi CD44 + T cells among CD8 − TCRβ + T cells in the inguinal dLNs of mice, 9 days p.i. ( n = 7 for Cd4 +/+ ; n = 10 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are mean ± SEM and are a summary of two independent experiments, *** p = 0.0007 (two-tailed Mann–Whitney U -test). e Representative FACS contour plot showing Tbet expression among CD4 + CD8 - TCRβ + T cells from the popliteal dLNs of mice, 28 days p.i. Data shown are representative of >3 independent experiments. f Proportion and numbers of Tbet+ cells among CD8 - TCRβ + CD11a hi CD44 + T cells from the popliteal dLNs of mice, 28 days p.i. ( n = 7 for Cd4 +/+ ; n = 10 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data shown are a summary of two independent experiments. *** p = 0.0001 (two-tailed Mann–Whitney U-test). g Numbers of Tbet+ cells among CD8 - TCRβ + CD11a hi CD44 + T cells from the popliteal dLNs of mice, 28 days p.i. ( n = 6/group) ** p = 0.0087 (two-tailed Mann–Whitney U-test). h Representative FACS contour plot showing GP 66–77 tetramer+ or CLIP-tetramer+ CD4 T cells (control) among splenocytes of LCMV-infected animals 8d p.i. i Quantification of data shown in h ( n = 5 for CLIP Tet + ; n = 10 for Cd4 +/+ ; n = 8 for Cd4 E4mΔ/Δ/ E4aΔ/Δ ). Data are representative of two independent experiments. ns not significant, p = 0.8098 (two-tailed Mann–Whitney U-test). j Gating strategy to detect GP 66–77 Tetramer+ cells among TCRβ + CD8- CD4 + and TCRβ + CD8- CD4- T cells. k Quantification of data shown in j ( n = 8). Data are representative of two independent experiments. *** p < 0.001 (two-tailed Mann–Whitney U-test).

    Techniques Used: Two Tailed Test, MANN-WHITNEY, Expressing, Infection

    a Venn diagram showing the number of genes that upregulate gene expression (a fold change >2 in gene expression from DN3 to CD4 + SP) intersected with genes that have 5hmC in either DP or CD4 + SP T cells (intragenic 5hmC log2 CMS-IP/input >2) intersected with genes containing open-chromatin ATAC-Seq peaks and H3K27Ac marks in either DP or CD4 + T cells. b Metascape bar graph showing top nonredundant enrichment clusters among genes that have novel-accessibility ATAC-Seq in activated T cells, undergo DNA demethylation, and upregulate RNA expression during thymic development. The number of genes in each cluster is indicated on the left and cluster IDs are shown on the right. Bar graph was generated using the Metascape gene annotation and analysis online resource. p -values were computed using hypergeometric test and Benjamini–Hochberg p -value correction algorithm as previously described in the publicly available Metascape interface . List of genes used for the enrichment analysis is found in Supplementary Table . c FACS plot showing CD4, ThPOK, CD5, and CD6 expression in CFSE-labeled T cells from control or Rorc(t) CreTg Tet1 fl/fl Tet2 fl/+ Tet3 fl/fl mice 72 hrs post activation. Naive CD4 T cells were FACS-sorted and activated with anti-CD3/CD28. Data is a representative of two experiments with at least two animals/genotype/experiment. d Cd4, Zbtb7b, Cd5 and Cd6 mRNA expression in control or Rorc(t) CreTg Tet1 fl/fl Tet2 fl/+ Tet3 fl/fl T cells activated in vitro for 96 hrs with anti-CD3/CD28. Data shown are mean ± SEM ( n = 3). p -values are indicated on graphs (unpaired two-tailed t -test). e IGV snapshots of the Zbtb7b locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC-Seq peaks in DP, CD4 + thymocytes, and Th0 CD4 + T cells. Red arrows denote currently undefined CREs. Green arrow shows the location of a previously validated CRE PE in CD4 + T cells , . f IGV snapshot of the Cd5 locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC Seq peaks in DP, CD4 + thymocytes and Th0 CD4 + T cells. Red arrows denote currently undefined CREs. g IGV snapshot of the Cd6 locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC-Seq peaks in DP, CD4 + thymocytes and activated CD4 + T cells. Red arrows denote currently undefined CREs.
    Figure Legend Snippet: a Venn diagram showing the number of genes that upregulate gene expression (a fold change >2 in gene expression from DN3 to CD4 + SP) intersected with genes that have 5hmC in either DP or CD4 + SP T cells (intragenic 5hmC log2 CMS-IP/input >2) intersected with genes containing open-chromatin ATAC-Seq peaks and H3K27Ac marks in either DP or CD4 + T cells. b Metascape bar graph showing top nonredundant enrichment clusters among genes that have novel-accessibility ATAC-Seq in activated T cells, undergo DNA demethylation, and upregulate RNA expression during thymic development. The number of genes in each cluster is indicated on the left and cluster IDs are shown on the right. Bar graph was generated using the Metascape gene annotation and analysis online resource. p -values were computed using hypergeometric test and Benjamini–Hochberg p -value correction algorithm as previously described in the publicly available Metascape interface . List of genes used for the enrichment analysis is found in Supplementary Table . c FACS plot showing CD4, ThPOK, CD5, and CD6 expression in CFSE-labeled T cells from control or Rorc(t) CreTg Tet1 fl/fl Tet2 fl/+ Tet3 fl/fl mice 72 hrs post activation. Naive CD4 T cells were FACS-sorted and activated with anti-CD3/CD28. Data is a representative of two experiments with at least two animals/genotype/experiment. d Cd4, Zbtb7b, Cd5 and Cd6 mRNA expression in control or Rorc(t) CreTg Tet1 fl/fl Tet2 fl/+ Tet3 fl/fl T cells activated in vitro for 96 hrs with anti-CD3/CD28. Data shown are mean ± SEM ( n = 3). p -values are indicated on graphs (unpaired two-tailed t -test). e IGV snapshots of the Zbtb7b locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC-Seq peaks in DP, CD4 + thymocytes, and Th0 CD4 + T cells. Red arrows denote currently undefined CREs. Green arrow shows the location of a previously validated CRE PE in CD4 + T cells , . f IGV snapshot of the Cd5 locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC Seq peaks in DP, CD4 + thymocytes and Th0 CD4 + T cells. Red arrows denote currently undefined CREs. g IGV snapshot of the Cd6 locus depicting p300 and H3K27Ac signals by ChIP-Seq in activated T (Th0) cells and ATAC-Seq peaks in DP, CD4 + thymocytes and activated CD4 + T cells. Red arrows denote currently undefined CREs.

    Techniques Used: Expressing, RNA Expression, Generated, Labeling, Activation Assay, In Vitro, Two Tailed Test, ChIP-sequencing

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    Thermo Fisher anti cd5
    Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show <t>CD5</t> versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.
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    Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show <t>CD5</t> versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.
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    a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, <t>CD5,</t> CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).
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    a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, <t>CD5,</t> CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).
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    a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, <t>CD5,</t> CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).
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    Image Search Results


    Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show CD5 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.

    Journal: eLife

    Article Title: A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage

    doi: 10.7554/eLife.80277

    Figure Lengend Snippet: Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show CD5 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.

    Article Snippet: Antibody , Anti-CD5 (rat monoclonal) , Thermo Fisher Scientific , Clone 53-7.3 , Conjugated to FITC (1/1000).

    Techniques: Staining, Expressing, Two Tailed Test, MANN-WHITNEY, Selection

    Journal: eLife

    Article Title: A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage

    doi: 10.7554/eLife.80277

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-CD5 (rat monoclonal) , Thermo Fisher Scientific , Clone 53-7.3 , Conjugated to FITC (1/1000).

    Techniques: Staining, Purification, Binding Assay, Magnetic Beads, Microscopy, Software

    a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, CD5, CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).

    Journal: bioRxiv

    Article Title: The Cul5 E3 Ligase Complex Is a Key Negative Feedback Regulator of TCR/IL2 Signaling and Anti-Tumor Activity in CD8 + T Cells

    doi: 10.1101/2022.11.16.516824

    Figure Lengend Snippet: a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, CD5, CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).

    Article Snippet: Antibodies used for flow cytometry are as follows: anti-mouse: Biolegend: CD8a-PE-Cy7 (Cat.100721), CD122-APC (Cat.105911), CD127-APC-Cy7 (Cat.135039), GZMB-APC (Cat.372203), IFNg-APC-Cy7 (Cat.505849), TNF-PE-Cy7 (Cat.506323), IL2-PB (Cat.503820), CD25-APC-Cy7 (Cat.101917), CD5-PB (Cat.100641), ICOS-APC (Cat.107711), PD1-PE-Cy7 (Cat.135215), CTLA4-APC (Cat.106309), CD62L-PE-Cy7 (Cat.104417), Vb5-PB (Cat.139515), Va2-PE (Cat.127807); Thermo Scientific: CD137-PB (Cat.48-1371-82); BD: CD107a-APC (Cat.560646), pSTAT5-Alexa 647 (Cat.562076), anti-human (Biolegend): GZMB-APC (Cat.372203), IFNg-PE (Cat.502508), CTLA4-APC (Cat.369611), anti-rabbit: IgG-PE (Thermo Scientific, Cat. P-2771MP), IgG-Alexa Fluor 350 (Thermo Scientific, Cat.A-11069), anti-pERK1/2 (Cell Signaling Technology, Cat. 9101), Rabbit polyclonal IgG anti-Cul5 (Thermo Scientific, Cat.A302-173A); SIINFEKL-H-2K(b) tetramer-BV421 (NIH Tetramer Core Facility, Cat.53995)

    Techniques: Mass Spectrometry, Flow Cytometry, Expressing, Activation Assay, Western Blot, Irradiation, Adoptive Transfer Assay