primary anti cd45 antibody  (Novus Biologicals)

Journal: The Journal of Clinical Investigation

Article Title: Immunotherapy targeting drug-tolerant Mycobacterium tuberculosis persisters accelerates tuberculosis cure in preclinical models

doi: 10.1172/JCI196648

Figure Lengend Snippet: ( A and B ) Representative lung sections (whole slides, rich DC–T cell fields of view and DC–T cell spatial plots) from RHZE alone ( A ) versus IN Mip3a/relMtb plus RHZE after 12 weeks of treatment ( B ) stained with antibodies for DAPI + only (dark blue, cell nuclei, top) or CD45 + (white, hematopoietic cells, bottom), CD45 + CD3 + (red, T cells, bottom), and CD45 + CD3 - CD11c + F4/80 – (yellow, DCs, bottom). Light brown arrows in low-magnification images (scale bars: 1 mm) indicate the specific areas shown in the corresponding high-magnification images (scale bars: 50 μm). Magenta arrows indicate DC–T cell colocalization. ( C and D ) Quantification of total DCls per DAPI + cells (percentage) and colocalization of DCs and T cells defined as the number of DCs within 10 μm of T cells (RHZE alone [ n = 6] vs. IN Mip3a/relMtb plus RHZE [ n = 4]). Assessment was performed on sections encompassing the entire left lung. * P < 0.05, by Mann-Whitney U test.

Article Snippet: Anti-CD45 primary antibody (1:200 dilution; Cell Signaling Technology, catalog 702575S) was applied at 36°C for 40 minutes.

Techniques: Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: An oncogenotype-immunophenotype paradigm governing the myeloid landscape in genetically engineered mouse models of prostate cancer

doi: 10.1101/2025.10.21.683668

Figure Lengend Snippet: A distinct myeloid cell distribution across different genotypes of tumor models. (A)Illustration for mouse models used in the study. (B) Kaplan-Meier plot of the survival analysis of four representative tumor models. (C) FACS analyses showing dichotomous infiltration of Ly6G + F4/80 − cells(neutrophils) and Ly6G−F480+ cells (macrophages) in four representative tumor models. Plots are gated on CD45 + CD11b+ cells. The experiments were repeated at least five times with similar results. (D) FACS analyses and quantification showing TIN percentage in the tumor microenvironment of vehicle- and SX-682-treated tumor-bearing mice. Plots are gated on CD45 + cells. (E) The bar plot of PS tumor weights of vehicle- and SX-682-treated tumor-bearing mice. (F) FACS analyses and quantification showing the percentage of CD3 + CD8 + T cells in the tumor microenvironment of vehicle- and SX-682-treated tumor-bearing mice. Plots are gated on CD45 + cells. (G) FACS analyses of T cell CFSE assay, showing the T cell proliferation after coculturing with TINs purified from PP, PS, PPS tumors. (H) Illustration of the central question: how tumor-intrinsic factors shape NES and MES TMEs to influence tumor progression. Statistical significance: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Unpaired nonparametric Mann Whitney test.

Article Snippet: Primary antibodies include anti-Mouse-CD45-FITC (Cytek Biosciences, 35-0451-U100, San Diego, CA, USA), anti-Mouse-CD11b-APC (Cytek Biosciences, 20-0112-U100, San Diego, CA, USA), anti-Mouse-Ly6G-PE-Cyanine7 (Cytek Biosciences, 60-1276-U100, San Diego, CA, USA), anti-Mouse-F4/80-PE (Cytek Biosciences, 50-4801-U100, San Diego, CA, USA), anti-Mouse-CD3-APC-Cyanine7 (Cytek Biosciences, 25-0032-U100, San Diego, CA, USA), anti-Mouse-CD8-APC (Cytek Biosciences, 20-0081-U100, San Diego, CA, USA), anti-Mouse-CD4-PE (Cytek Biosciences, 50-0041-U100, San Diego, CA, USA).

Techniques: CFSE Assay, Purification, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Clinical and molecular dissection of CAR T cell resistance in pancreatic cancer

doi: 10.1016/j.xcrm.2025.102301

Figure Lengend Snippet: Locally delivered huCART-meso cells are enriched for GzmK + Tem phenotypes and exhibit a dysfunctional T cell phenotype including upregulation of SOX4 and ID3 (A) Summary of the samples collected for flow cytometry and scRNA-seq analyses. (B) Representative flow cytometry plot of patient 04 showing detection of peritoneal fluid infiltrating CD3 + cells with surface expression of the huCART-meso. (C and D) CAR detection performed by qPCR on peripheral blood (left) and peritoneal fluid (right) of patient 04 (C) and patient 05 (D). (E) UMAP projection of scRNA-seq data from sorted CD3 + CD45 + cells infiltrating the peritoneal fluid 7 days post intraperitoneal infusion (patient 04) or 26 days post infusion (patient 05). UMAP plot is labeled with T cell subsets. (F) huCART-meso expression associated with (E). (G) Distribution of huCAR-meso+ (CARpos) and endogenous CAR-negative T cells (CARneg) across cell type clusters defined in (E). Unknown cluster from (E) was excluded. The y axis represents the percent of cells found within each cell type cluster, calculated by taking the number of cells per condition (condition being CARpos or CARneg) within each cell type cluster and dividing by the total number of cells in that condition, calculated for each patient individually. Dots represent the individual patient values, and the bar plot represents the average of the patient values; moderated t test via the propeller method ( n = 2 patients). p values denoted as ∗ p < 0.05 and ∗∗ p < 0.01. (H) Expression of the 30-gene CAR T dysfunction signature in CD3 + CARpos and CARneg T cells of patients 04 and 05 post infusion. (I) Gene expression levels of huCART-meso, SOX4, ID3, SRGAP3, NDFIP2, CTLA4, TNFRSF9, PDCD1, HAVCR2, and TIGIT in CARpos (left, red) and CARneg cells (right, green). CARneg cells were randomly downsampled to have an equal number of CARpos and CARneg cells for analyses in (G)–(I). See also .

Article Snippet: Rabbit monoclonal primary antibody against human CD45 (CD45-LCA; Cell Signaling Technology, catalog no. 13917) was used at concentrations of 1:300 and incubated on the sections for 45 min at room temperature.

Techniques: Flow Cytometry, Expressing, Labeling, Gene Expression

Journal: Cell Reports Medicine

Article Title: Clinical and molecular dissection of CAR T cell resistance in pancreatic cancer

doi: 10.1016/j.xcrm.2025.102301

Figure Lengend Snippet: Mice injected with single KO of SOX4 or ID3 eventually experience tumor recurrence (A) Experimental design for recurrent tumor model. (B) KO efficiency in sorted T cells from relapsing tumors. Each dot represents a technical replicate (independent sequencing for KO assessment from one mouse per group); mean ± SEM. (C) CD3 + CD45 + T cell infiltration in ND561 WT and SOX4KO relapsing tumors (left) and percent of CD3 + CD45 + surface huCART-meso in relapsing tumors and infused products (right). Each dot represents a technical replicate ( n = 1 mouse per group); Mann-Whitney U test (left) and two-Way ANOVA and Sidak post hoc test (right); mean ± SEM. (D) Total number of MSLN + Epcam + cells (#) per gram of tumor (left) and representative flow cytometry plots (right) of MSLN expression in ND561 WT and SOX4KO-treated relapsing tumors ( n = 1 mouse per group). AsPC-1 cells were used as positive control and MSLN minus as negative control for MSLN staining; Mann-Whitney U test. Error bars represent variation between technical replicates; mean ± SEM. (E) UMAP projection of scRNA-seq data from recurrent TILs harvested from mice injected with WT CAR T cells (ND561, n = 1 donor) and SOX4-KO CAR T cells (ND561 and ND539, n = 2 donors). (F) UMAP projection with broad T cell subsets labeled. NK-like T cells clustered on the left (cluster 2) and all other clusters were defined as GZMK + exhausted T cells (Tex), right. (G) huCART-meso expression in recurrent tumor TILs. (H) Cluster distribution depicting the frequency that WT and SOX4KO-injected recurrent tumor TILs were found within the broad clusters defined in (F). y axis represents the percent of cells found within each cell type cluster for each condition (WT or SOX4KO). The percent was calculated by taking the number of cells per condition within each cell type cluster and dividing by the total number of cells in that condition. SOX4KO condition was averaged for the two mice and visualized via bar plot, and circles depict the individual values for each mouse; moderated t test using the propeller method. (I) Gene expression levels of TNFRSF9, TIGIT, LAG3, and HAVCR2 in recurrent tumor CARpos TILs extracted from mice injected with WT and SOX4KO CAR T cells. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ns: not significant. See also .

Article Snippet: Rabbit monoclonal primary antibody against human CD45 (CD45-LCA; Cell Signaling Technology, catalog no. 13917) was used at concentrations of 1:300 and incubated on the sections for 45 min at room temperature.

Techniques: Injection, Sequencing, MANN-WHITNEY, Flow Cytometry, Expressing, Positive Control, Negative Control, Staining, Labeling, Gene Expression

Journal: bioRxiv

Article Title: SMART: A Spatio-Molecular Atlas of Response Trajectories in Triple-Negative Breast Cancer

doi: 10.1101/2025.08.27.672673

Figure Lengend Snippet: Multimodal, longitudinal profiling of triple-negative breast cancer (TNBC). Tissue biopsies were obtained before, during, and after neoadjuvant chemotherapy (NACT) from responder (R) and non-responder (NR) TNBC patients (black frame). Three serial sections were taken from each sample and subjected to distinct analytical modalities. Section 1 was stained with H&E (pink frame), imaged, and annotated by a pathologist to identify histological features and quantify stromal tumour-infiltrating lymphocytes (sTILs). Section 2 underwent spatial transcriptomics (ST) profiling (red frame) using the NanoString/Bruker GeoMx Digital Spatial Profiler (DSP) to capture RNA transcripts from epithelial (PanCK+) and immune (CD45+) cells for downstream analysis. Immunofluorescent (IF) images for each AOI were obtained from the GeoMx DSP (yellow frame) and subjected to downstream imagebased network analysis. Section 3 was analysed using imaging mass cytometry (IMC) (green frame) to quantify the expression of 35 protein markers. The methods toolbox (blue frame) displays all analytical tools developed in this study. The SMART dataset compiles all datasets and analytical methods which are hosted on PharosAI and made publicly available (See Data Availability).

Article Snippet: First, primary anti-CD45 antibody (2B11 with PD7/26) [ Novus Biologicals; #NBP2-34528AF594 ] at a concentration of 5 μg μL −1 and primary anti-Ki67 antibody (D2H10) [ Cell Signalling; #44092 ] at 6 μg μL −1 .

Techniques: Staining, Imaging, Mass Cytometry, Expressing

Journal: bioRxiv

Article Title: SMART: A Spatio-Molecular Atlas of Response Trajectories in Triple-Negative Breast Cancer

doi: 10.1101/2025.08.27.672673

Figure Lengend Snippet: A) Dot plot indicating the relative proportion of immune cell subsets per TIME cluster, determined by consensus clustering with k = 7. B) Horizontal stacked bar charts showing the frequency of TIME states per pre-NACT sample from responders (n = 45, left panel), non-responders (n = 30, middle panel) and post-NACT samples from nonresponders (n = 36, right panel). C) Horizontal stacked bar charts indicating changes in TIME distribution in patient-matched pre-NACT and post-NACT samples, where each row indicates one patient. D) Patient-matched dot plots indicating the change in TIMEs frequencies from pre-NACT to post-NACT samples. Paired wilcoxon test p -values are given above the plot, significance was considered as p < 0.05. E) Forest plot showing the odds ratio (OR), 5% and 95% confidence intervals and p -values for TIME frequencies associated with responses to NACT. F) Barcharts depicting the relative frequencies of TIME1 (top panel), TIME2 (middle panel) and TIME3 (bottom panel) in pre-NACT samples between responders and non-responders (left panel) and between patients with high sTILs ( > 30%) versus low sTILs ( < 30%)(right panel). Wilcoxon test p -values are provided within the plot, significance was defined as p < 0.05. G) Boxplot indicating the TLS score of CD45+ AOIs in each TIME. Pairwise comparisons between TIMEs were performed with p-values adjusted for multiple testing using the Benjamini-Hochberg (BH) method. Statistical significance is denoted as follows: p < 0.001 (***), p < 0.01 (**), p < 0.05 (*). Asterisks correspond to adjusted p −values. H) Heatmap displaying the relative expression of each gene from the TLS signature (reference is in the overleaf version) in CD45+ AOIs in TIME3, cut to three trees. The relative expression is given on a colour scale ranging from blue (low expression) to red (high expression). The green box indicates a cluster of TIME3 AOIs with high expression of nearly all genes in the TLS signature. I) Representative GeoMx IF (top panel) and matched H&E (bottom panel) images corresponding to TIME3 AOIs identified in panel G (far-right tree, green box). GeoMx IF images visualise DNA (blue), PanCK+ cells (green) and CD45+ cells (red) identifying the nuclei, epithelial cells and immune cells, respectively.

Article Snippet: First, primary anti-CD45 antibody (2B11 with PD7/26) [ Novus Biologicals; #NBP2-34528AF594 ] at a concentration of 5 μg μL −1 and primary anti-Ki67 antibody (D2H10) [ Cell Signalling; #44092 ] at 6 μg μL −1 .

Techniques: Expressing

Journal: bioRxiv

Article Title: SMART: A Spatio-Molecular Atlas of Response Trajectories in Triple-Negative Breast Cancer

doi: 10.1101/2025.08.27.672673

Figure Lengend Snippet: Boxplots indicate the relative proportion of immune cell subtypes in A) TIME3 AOIs co-localised with EA4 and B) TIME4 AOIs co-localised with EA4, between responders and non-responders. Wilcoxon test p -values are given above the plot, significance was defined as p < 0.05. The total number of patients is given below the plot. C) Dot plot depicts the correlation between tumour-derived chemokines expressed in EA4 with the proportions of immune cell subtypes in the corresponding TIME3 or TIME4 AOIs. Statistical significance was considered when p¡0.05 and is indicated by a thick black outline. D) Dotplot indicates the Pearson correlation between the relative proportions of pDCs and activation of pathways related to B-cell and CD8+ T cell stimulation within TIME3 (left panel) or TIME4 (right) AOIs when co-localised with EA4, in responders compared to non-responders. Statistical significant was considered when p < 0.05 and is indicated by a thick black outline. E) Schematic depicting the mean distance between epithelial (green) and immune (red) cells. F) Boxplot comparing the average distance between tumour (PanCK+) and immune (CD45+) cells in ROIs with EA4 localised with TIME4. Wilcoxon t-test was used to compute statistical significance. G) Dotplot indicates the correlation between the average distance between tumour (PanCK+) and immune (CD45+) cells and the relative enrichment of pathways related to antigen processing and presentation by antigen presenting cells (APCs) in TIME3 or TIME4 AOIs when localised with EA4. Statistical significant was considered when p < 0.05 and is indicated by a thick black outline.

Article Snippet: First, primary anti-CD45 antibody (2B11 with PD7/26) [ Novus Biologicals; #NBP2-34528AF594 ] at a concentration of 5 μg μL −1 and primary anti-Ki67 antibody (D2H10) [ Cell Signalling; #44092 ] at 6 μg μL −1 .

Techniques: Derivative Assay, Activation Assay, Cell Stimulation