flex polyclonal rabbit anti-human cd3 antibody  (Agilent technologies)

Journal: Nature Cancer

Article Title: The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2

doi: 10.1038/s43018-023-00712-x

Figure Lengend Snippet: (a) Quantification and representative images of Panc02 WT and PD-L2-KO tumors in nude mice, untreated and treated with doxorubicin on days 7 and 10. Luminescence units are photon/sec/cm 2 /stereoradian in the images. N = 3 for PD-L2-WT + PBS mice, n = 5 for PD-L2-KO + PBS, n = 6 for both doxo-treated groups. (b) Gating strategy to define circulating CD4 + and CD8 + T cell populations. (c) Percentage of CD4 + and CD8 + T cells among total CD45 + CD3 + cells in blood of mice after treatment with blocking anti-CD4 and anti-CD8 antibodies. N = 6-7 mice. Data are presented as mean ± SEM.

Article Snippet: Unspecific unions were blocked using 3% goat normal serum (catalog no. 16210064, Thermo Fisher Scientific) for 60 min. Primary antibody FLEX Polyclonal Rabbit Anti-Human CD3 (catalog no. IR503, Agilent Technologies) was diluted 1:10 with Dako antibody diluent (code no. S0809, Agilent Technologies) for 120 min at room temperature.

Techniques: Blocking Assay

Journal: Nature Cancer

Article Title: The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2

doi: 10.1038/s43018-023-00712-x

Figure Lengend Snippet: (a) Percentage of NK cells (NK1.1 + ), macrophages (CD11b + Gr1 - ) and lymphocytes (CD3 + TCRb + ) relative to total CD45 + cells, in WT and PD-L2-KO tumors untreated or treated with doxorubicin at days 7 and 10, analysed by mass cytometry. N = 4 mice for all conditions. (b) Percentage of PD-1 + cells among subsets of infiltrating T cells, analysed by mass cytometry. N = 3 mice for WT + PBS and PD-L2-KO + doxo, n = 4 for WT + doxo and PD-L2-KO + PBS (c) Quantification of tumor infiltrating CD3 + lymphocytes in WT and PD-L2-KO tumors, untreated or treated with doxorubicin, analysed by immunohistochemistry. N = 4 for PBS-treated groups and n = 5 for doxo-treated groups. None of the changes were significant (1 way ANOVA, Tukey post-test). Data are presented as mean ± SEM. (d) Representative Gr1 stainings in sections of PD-L2-KO tumors treated with doxorubicin and subject to depletion of CD4 + (n = 8) or CD8 + (n = 9) T cells, as well as IgG-treated controls (n = 6) from Fig. .

Article Snippet: Unspecific unions were blocked using 3% goat normal serum (catalog no. 16210064, Thermo Fisher Scientific) for 60 min. Primary antibody FLEX Polyclonal Rabbit Anti-Human CD3 (catalog no. IR503, Agilent Technologies) was diluted 1:10 with Dako antibody diluent (code no. S0809, Agilent Technologies) for 120 min at room temperature.

Techniques: Mass Cytometry, Immunohistochemistry

Journal: Nature Cancer

Article Title: The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2

doi: 10.1038/s43018-023-00712-x

Figure Lengend Snippet: a , Tumor growth in PyMT mice treated weekly (from week 9 until week 12) with αPD-L2 alone (TY25, 10 mg kg −1 ) or the same dose of isotype control every 3 days, doxorubicin alone or a combination of both as indicated. αPD-L2 monotherapy ( n = 3 mice); PBS and doxorubicin + IgG groups ( n = 4); doxorubicin + αPD-L2 ( n = 5). Two-way ANOVA. Inverted red triangles indicate day of doxorubicin treatment. b , c , SA-β-gal, CD3, CD4 and CD8 staining of untreated tumors and tumors treated with doxorubicin alone or in combination with αPD-L2 blocking antibody (TY25, 10 mg kg −1 ) or the same dose of isotype control ( b ), as in a , and analyzed at week 13 ( c ). Vehicle-treated group ( n = 4); doxorubicin-treated groups ( n = 5). Representative images are shown. Statistical significance shown versus the rest of the experimental conditions. One-way ANOVA with Tukey post-hoc test. Scale bar, 100 μm. d , SA-β-gal and PD-L2 costaining in tumor samples from PyMT mice treated weekly with doxorubicin (4 mg kg −1 ) at postnatal weeks 9–12. The indicated groups were treated with αPD-L2 (TY25, 10 mg kg −1 ), or the same dose of IgG isotype control, every 3 days. Representative images and quantification are shown. Samples were obtained at postnatal week 13 (that is, 7 days after the last doxorubicin dose). Vehicle-treated group ( n = 4 mice); doxorubicin-treated groups ( n = 5 mice). A one-way ANOVA was used for significant changes versus the rest of the conditions. Scale bar, 100 μm. e , SA-β-gal staining, p21 mRNA levels, PD-L2 mRNA levels and PD-L2 protein levels measured using fluorescence-activated cell sorting (FACS), with and without bleomycin treatment, in human head and neck cancer primary culture from patient VHIO-008. Gene expression ( n = 4 independent replicates); FACS ( n = 1). Scale bar, 50 μm. Two-sided t -tests were used.

Article Snippet: Unspecific unions were blocked using 3% goat normal serum (catalog no. 16210064, Thermo Fisher Scientific) for 60 min. Primary antibody FLEX Polyclonal Rabbit Anti-Human CD3 (catalog no. IR503, Agilent Technologies) was diluted 1:10 with Dako antibody diluent (code no. S0809, Agilent Technologies) for 120 min at room temperature.

Techniques: Staining, Blocking Assay, Fluorescence, FACS, Expressing

Journal: Frontiers in Oncology

Article Title: Desensitization of Capsaicin-Sensitive Afferents Accelerates Early Tumor Growth via Increased Vascular Leakage in a Murine Model of Triple Negative Breast Cancer

doi: 10.3389/fonc.2021.685297

Figure Lengend Snippet: No significant difference in the CD3 immunoreactivity-based inflammatory response in the area surrounding the 4T1 tumors after RTX-pretreatment. (A) Ratio of the inflammatory ring area to the entire tumor area (B) Representative anti-CD3 stained sections, demonstrating tumor annotation (red) and the inflammatory ring (blue) with low (1.4x) magnification. (C) CD3 expression measured by quantitative immunohistochemistry (relative mask area %). (D) Representative anti-CD3 stained slides with high (23x) magnification. The inflammatory ring is marked with blue annotation.

Article Snippet: All incubations were set to room temperature with the samples washed between incubations in TBST buffer for 2 x 5 min. H&E, CD3 (FLEX Polyclonal Rabbit Anti-Human CD3 RTU, produced by DAKO), CD31 (clone D8V9E, produced by Cell Signaling Technology) and CD45-immunostained (clone M0701, produced by DAKO) slides were digitalized by Pannoramic Digital Slide Scanner (3DHISTECH Ltd., Budapest, Hungary).

Techniques: Staining, Expressing, Immunohistochemistry