Journal: Cell Reports Medicine

Article Title: Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in DSRCTs

doi: 10.1016/j.xcrm.2024.101582

Figure Lengend Snippet: ScRNA-seq recapitulates DSRCT cellular composition (A) DSRCT bulk and single-cell multiomic profiling. (B) Patient and sample characteristics. L1: one prior treatment line; L2: two prior treatment lines. (C) Uniform Manifold Approximation and Projection (UMAP) showing Int_sc dataset sample-of-origin. (D) UMAP showing DSRCT signature score. (E) UMAP showing DSRCT neotranscripts expression. (F) UMAP (left panel) and barplot (right panel) highlighting DSRCT cell subpopulations. (G) Heatmap showing expression Z - score of the top 50 DEGs of Int_sc cell types. (H) H&E and IHC stainings for THY1, CD68/CD163, and CD3. The scale bar is displayed in the bottom left corner of each panel, representing 1 mm in the top panel and 200 µm in the four lower panels. (I) DSRCT bulk RNA-seq cell subpopulation deconvolution.

Article Snippet: Polyclonal rabbit anti-human CD3 , Dako (Agilent) , Cat# A0452; RRID: AB_2335677.

Techniques: Expressing, RNA Sequencing Assay

Journal: Cell Reports Medicine

Article Title: Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in DSRCTs

doi: 10.1016/j.xcrm.2024.101582

Figure Lengend Snippet:

Article Snippet: Polyclonal rabbit anti-human CD3 , Dako (Agilent) , Cat# A0452; RRID: AB_2335677.

Techniques: Diagnostic Assay, Recombinant, Red Blood Cell Lysis, Ligation, Protease Inhibitor, Immunoprecipitation, Staining, Fluorescence, Expressing, Purification, Isolation, Control, Software, Microscopy, Imaging

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: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: Examples of the 500 × 500 pixel CD3 IHC stained images used in the experiments. There were 20 such images. The brown pixels indicate CD3+ T cells while the blue pixels are the CD3− T cells. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques: Staining

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: Flowchart of the CD3+ and CD3− T cells segmentation method.

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques:

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: Scatter plots of two sample CD3+ stained images (a) in (b) RGB and (c) L*a*b* color formats. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques: Staining

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: An example showing (a) a sample image decomposed into (b) CD3+ T cell regions detected by the highlighted in red, (c) CD3− T cell regions highlighted in blue and (d) background regions in white. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques:

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: An evaluation of (a) sensitivity and (b) specificity of CD3+ and CD3− cell segmentation for all four pathologists

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques:

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: Results of computerized CD3+ T cell quantification [based on Eq. (3)] shown against the four pathologists’ quantification estimates. The computerized quantification consistently results within 95% confidence interval of the marked CD3+ T cell population of the four pathologists as shown in (a). It is also observed that the four pathologists exhibit a consistent and predictable pattern in marking CD3+ and CD3− T cells. A different pattern is seen in (b) which shows results of CD3+ T cell population estimation through “eyeballing.” For visual clarity, both data in (a) and (b) are sorted in ascending order to the mean percentage of CD3+ T cell population in (a). [Color figure can be viewed at wileyonlinelibrary.com.]

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques:

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: Performance of pathologists and the computer in determining CD3+ T cell coverage

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques:

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Computer-assisted Quantification of CD3+ T Cells in Follicular Lymphoma

doi: 10.1002/cyto.a.23049

Figure Lengend Snippet: The Bland-Altman plots illustrating the relationships between the “eyeballing” CD3+ T cells estimates and the average cell marking measurements for all four pathologists shown in (a) through (d). The computerized quantification is compared to the average cell marking measurement and shown in (e). The plots represent bias versus (pA +pn)/2. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Polyclonal rabbit anti-human CD3 antibody was used from Dako A0452 at 1:400 dilution.

Techniques: