Journal: Journal of Nanobiotechnology

Article Title: Targeting CD39 boosts PD-1 blockade antitumor therapeutic efficacy via strengthening CD8 + TILs function and recruiting B cells in cervical cancer

doi: 10.1186/s12951-025-03500-0

Figure Lengend Snippet: CD39 inhibitor combined with PD-1 blockade improved therapeutic efficacy by promoting CXCL13 release and B cell activation . ( A ) Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of CXCL13 in the serum samples of isotype, αPD-1, CD39i, and combined treatment groups of mice. ( B ) The content of CXCL13 in murine tumors was detected using ELISA. ( C ) Flow cytometry allowed detection of the proportion of CXCR5 + B cells in murine tumors. ( D - E ) Flow cytometry ( D ) and dot plots ( E ) showed the proportion of activated B cell infiltration in murine tumors. ( F ) The dot plot displayed the proportion of GCB cells in tumor B220 + B cells. ( G , H ) Dot plots showed the proportion of dark zone (DZ) ( G ) and light zone (LZ) ( H ) in GCB cells in mouse tumors. ( I , J ) Representative histograms ( I ) and summarized Mean Fluorescence Intensity (MFI) diagram ( J ) showed the expression of BCL6 in GCB cells. ( K ) The dot plot showed the proportion of plasma cells among CD19 + B cells of mouse tumors. ( L ) The dot plot displayed the proportion of memory B cells in mouse tumor B220 + B cells. The values were obtained using the Kruskal–Wallis test. * , P < 0.05; ** , P < 0.01; ***; P < 0.001; ns , not significant . Abbreviations: GCB, geminal center B cells; DZ, dark zone; LZ, light zone

Article Snippet: The sections were stained with a CD19 antibody (27949-1-AP; Proteintech; 1:500), Ki67 antibody(ab16667;Abcam;1:200), TUNEL antibody(G1507; Servicebio; 1:200) followed by a secondary antibody (GB23303; Servicebio; 1:200).

Techniques: Drug discovery, Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Expressing, Clinical Proteomics

Journal: Journal of Nanobiotechnology

Article Title: Inverse-designed plasmonic biosensors with LSPR-SPP-wood anomaly coupling enhanced for biomolecular analysis

doi: 10.1186/s12951-025-03930-w

Figure Lengend Snippet: High-throughput affinity sensing platform for ADC drug development. ( a ) The association process of incubating PD-1 on the chip to detect Sintilimab. ( b ) The long-time dissociation process of PD-1 with Sintilimab. ( c ) Dynamic real-time and fitting curves of the association and dissociation phases of the interaction between PD-1 and Sintilimab. ( d ) Schematic diagram of the high-throughput antibody affinity detection protocol, with each group, repeated three times in the three samples tested, and the concentration of A-H in each column decreasing incrementally. ( e ) Schematic diagram of the main events in the antibody drug fishing and affinity detection process. ( f-h ) Dynamic real-time and fitting curves of the association and dissociation phases for Polatuzumab with CD79B, Adalimumab with TNF-α, and Sacituzumab govitecan with TROP2, detected using the unoptimized sensor. ( i-k ) Dynamic real-time and fitting curves of the association and dissociation phases for Polatuzumab with CD79B, Adalimumab with TNF-α, and Sacituzumab govitecan with TROP2, detected using the optimized IDMM-SPR sensor

Article Snippet: Bovine serum albumin (BSA), Phosphate-buffered saline (PBST), 11-mercaptoundecanoic acid (MUA), 1-ethyl3-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Dimethyl Sulfoxide (DMSO), Fish gelatin,, ethanolamine, and phosphate-buffered saline (PBS) buffer, Polyethylene glycol 2000 (PEG) were purchased from Sigma-Aldrich; Protein A, IgG, CRP, and CRP ab, hTGFBR1 were purchased from monoclonal anti-CRP capture and detection antibodies were purchased from Beijing Sino Biological, Inc. (Beijing, China); Sintilimab, Adalimumab, Polatuzumab, Sacituzumab, PD-1, TNF-α, CD79B, TROP2, Quercetin were purchased from MedChemExpress.

Techniques: High Throughput Screening Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: CD11b is required for stretch-mediated changes in macrophage activation. (A) Representative Western blot of CD11b and GAPDH for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0% and 20% static and cyclic stretch (left). Quantification of average across three independent experiments for CD11b expression (right). Values were normalized to GAPDH and made relative to 0% stretch and unstimulated condition. (B) Representative immunofluorescence images (top) and quantification of relative Itgam gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Data relative to siControl condition. (C) Secretion of TNFα, IL6, and MCP1 for unstimulated and IFNγ/LPS stimulated macrophages treated with siControl or siCD11b and exposed to either 0% control, 20% static, or 20% cyclic stretch. Data normalized to a siControl and IFNγ/LPS treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Arg1 gene expression in IL4/IL13 stimulated and siControl or siCD11b treated BMDMs exposed to 0% control, 20% static, or 20% cyclic stretch. Data relative to 0% siControl condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (A, C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Gene Expression, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Modulation of CD11b by adhesion time regulates stretch-mediated macrophage inflammatory responses. (A) Phase contrast images (top) of macrophages following 4 hrs (left) and 24 hrs (right) of adhesion prior to stimulation and stretch. Fluorescence images (bottom) of macrophages labelled for CD11b (red), actin (green), and nuclei (blue) following 4 hrs (left) and 24 hrs (right) of culture. (B) Averaged relative median fluorescence intensity across three independent experiments of CD11b as measured by flow cytometry. Values normalized to 4 hrs adhesion condition. (C) Secretion of TNFα for unstimulated and IFNγ/LPS stimulated macrophages exposed to 0% and 20% cyclic stretch after 4 hrs (left) and 24 hrs (right) of adhesion. Values are normalized to a 0% stretch IFNγ/LPS internal control within each biological replicate. (D) Representative Western blots (left) and corresponding quantification for ARG1 (right) for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages allowed to adhere for 4 hrs prior to stimulation and stretch. Values were normalized to GAPDH and made relative to IL4/IL13 stimulated and 0% stretch conditions, respectively. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Fluorescence, Flow Cytometry, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Crosstalk between Piezo1 and CD11b mediates macrophage response to stretch. (A) Representative immunofluorescence images (left) and quantification of mean Piezo1-tdT intensity in unstimulated, IFNγ/LPS, and IL4/IL13 treated macrophages exposed to 0% control, 20% static, or 20% cyclic stretch. Data normalized to the 0% control condition. (B) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with siControl or siPiezo1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a siControl treated internal control exposed to 0% stretch within each biological replicate. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or Yoda1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a DMSO treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Piezo1 gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Gene expression is normalized to the siControl treated condition. (E) Relative Itgam, Itgb1, Itgb2 , and Itgb3 gene expression in unstimulated and siControl or siPiezo1 treated macrophages. Gene expression is normalized to the siControl treated condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Immunofluorescence, Control, Gene Expression, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Stretch-induced changes in macrophage activation require modulation of actin. (A) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0%, 20% static, and 20% cyclic stretch. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Data normalized to the unstimulated and 0% stretch control. (B) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to siControl or CD11b siRNA. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Values normalized to unstimulated and siControl condition. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Values are normalized to a DMSO, 0% stretch, and IFNγ/LPS stimulated internal control within each biological replicate. (D) Representative Western blot (left) and quantification of ARG1 expression in IL4/IL13 stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Expression is relative to GAPDH. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by paired t-test (A, B) and Student’s t-test (C, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Fluorescence, Control, Western Blot, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Summary of stretch mediated changes in macrophage function.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Expressing

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: The target cells for training immunity induced by KK2DP7 are cells in the non‐lymphocyte layer of the spleen. a) Validation flowchart for KK2DP7‐induced training immunity in spleen target cell population. b) Cells in the splenic lymphocyte layer of mice trained with KK2DP7 and stimulated with LPS show no significant changes in the secretion of training‐immunity‐associated TNF‐α, IL‐1β, IL‐6, and IFN‐γ (n = 3). c) Cells in the splenic non‐lymphocyte layer of mice trained with KK2DP7 and stimulated with LPS exhibited a significant upregulation in the secretion of training‐immunity‐associated TNF‐α, IL‐1β, IL‐6, and IFN‐γ (n = 3). d) Representative flow cytometry plots of target cells in the non‐lymphocytic layer of mouse spleen are presented. e) TNF‐α production by cells in the non‐lymphocytic layer of the mouse spleen was significantly elevated after training with KK2DP7 and stimulation with LPS, whereas no changes in TNF‐α production were observed in cells in the lymphocytic layer (n = 3). f) TNF‐α production by CD45 + cells in the non‐lymphocytic layer of the mouse spleen was significantly elevated after training with KK2DP7 and stimulation with LPS, whereas no changes in TNF‐α production were observed in CD45 ‐ cells (n = 3). g) Following training with KK2DP7 and LPS stimulation, the proportion of CD45 + CD11b + cells in the non‐lymphocyte layer of the mouse spleen was found to be significantly upregulated (n = 3). h–k) Training with KK2DP7 and LPS stimulation led to a significant increase in the proportion of macrophages (CD11b + F4/80 + ), neutrophils (CD11b + Ly6G + ), monocytes (CD11b + Ly6C + ), and dendritic cells (CD11b + CD11c + ) expressing TNF‐α in the non‐lymphocytic layer of the mouse spleen (n = 3). All values presented in this figure are expressed as the mean ± S.D., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Biomarker Discovery, Flow Cytometry, Expressing, Comparison

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: KK2DP7 induces the training of immunotherapy against tumors by activating CD11b + cells in the non‐lymphocytic layer of the spleen. a) The validation flowchart for KK2DP7‐induced training immunity in the spleen target cell population is presented below. b) CD11b + cells in the splenic non‐lymphocytic layer of mice trained with KK2DP7 and stimulated with LPS exhibited a significant upregulation of TNF‐α, IL‐1β, IL‐6, and IFN‐γ secretion, which is indicative of training immunity (n = 3). c) CD11b ‐ cells in the splenic nonlymphocytic layer of mice trained with KK2DP7 and stimulated with LPS did not exhibit any significant changes in the secretion of training‐immunity‐associated TNF‐α, IL‐1β, IL‐6, and IFN‐γ (n = 3). d,e) The CD11b + cells in the non‐lymphocyte layer of the spleen, which were trained with KK2DP7 and stimulated by tumor cells or tumor cell culture supernatant, demonstrated a significant upregulation of immune‐related TNF‐α, IL‐1β, IL‐6, and IFN‐γ secretion (n = 3). f) A flowchart of the adoptive transfer of CD11b + cells from the non‐lymphocyte layer of the spleen, following training with KK2DP7, for antitumor application is provided. g) The average tumor growth curve of mice in different groups is shown (n = 6). h) The survival rates of mice in different groups are presented (n = 6). i–k) Individual tumor growth curves from g are displayed. All values presented in this figure are expressed as the mean ± S.D., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. Survival curves were obtained using the Kaplan–Meier method and compared by the log‐rank test. **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Biomarker Discovery, Cell Culture, Adoptive Transfer Assay, Comparison

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: Significant differences were observed in the transcriptome and ATAC sequencing of CD11b + cells in the non‐lymphocyte layer of the mouse spleen following KK2DP7 treatment. a) Flowchart illustrating the target cell acquisition process for sequencing. b) Differential gene expression in CD11b + cells pretreated with KK2DP7 compared with those from PBS‐treated mice in RNA‐sequence (n=3). A volcano plot is presented, displaying the distribution of the adjusted p values (log10(padj)) and fold changes (log2 fold change). c) A heatmap of genes from KK2DP7‐treated mice as compared with PBS‐treated mice in RNA‐sequence is also included. d) An interaction network diagram of all changing genes in b is provided, as well as e) a differential peak expression in mice pre‐treated with KK2DP7 compared with from PBS‐treated mice in ATAC‐sequence. A volcano plot is presented, which shows the distribution of the adjusted p values (log10(padj)) and fold changes (log2 fold change). f) A heatmap is also provided, which shows the genes from KK2DP7‐treated mice as compared with PBS‐treated mice in ATAC sequence analysis. g) Gene ontology (GO) enrichment of immune‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in assay for RNA‐seq analysis. h) Gene ontology (GO) enrichment of immune‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in assay for transposase‐accessible chromatin sequencing (ATAC‐seq). i) Among the genes with shared changes in both RNA‐seq and ATAC‐seq, GO enrichment of immune‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice is analyzed. j) An expression heatmap of immune‐related genes that are commonly varied in both RNA‐seq and ATAC‐seq is provided. k) An interaction network diagram of all genes shown in j is included.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Sequencing, Gene Expression, Expressing, RNA Sequencing

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: The proliferation, phagocytosis and cytotoxicity of CD11b + cells in the non‐lymphocytic layer of the spleen are significantly enhanced following KK2DP7 training. a) Schematic diagram illustrating validated cell functions. b‐c) Gene Ontology (GO) enrichment analysis of cell proliferation‐related pathways in mice treated with KK2DP7 revealed a significant upregulation compared to mice treated with PBS, as evidenced by RNA and ATAC sequencing. d‐e) The expression heatmap of cell proliferation‐related genes and the interaction network diagram of all genes in the heatmap demonstrated a clear distinction between the two groups. f) The results of flow cytometry demonstrated a significant increase in the proliferation of CD11b + cells in the non‐lymphocyte layer of the spleen following KK2DP7 training and secondary stimulation. g‐h) GO enrichment of cell endocytosis‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in RNA and ATAC sequencing. i) Expression heatmap of cell endocytosis‐related genes. j) Network diagram of all genes in i. k) GO enrichment of cell cytotoxicity‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in RNA sequencing. l) Expression heatmap of cell cytotoxicity‐related genes. m) Network diagram of all genes in l. n) The results of flow cytometry demonstrated that following KK2DP7 training, the capacity of CD11b + cells in the non‐lymphocyte layer of the spleen to phagocytose tumor cells was significantly enhanced (n=3). o) The results of flow cytometry indicated that the capacity of CD11b + cells in the non‐lymphocyte layer of the spleen to kill tumors was significantly enhanced following KK2DP7 training (n=3). All values presented in this figure are expressed as the mean ± s.d., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. In contrast, a student's t‐test was utilized for two‐group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Sequencing, Expressing, Flow Cytometry, RNA Sequencing, Comparison

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: Transcriptome changes and epigenetic reprogramming induced by immune training in KK2DP7. a) Pathways of KEGG enrichment in CD11b + cells of the non‐lymphocytic layer of the spleen in mice treated with KK2DP7. b) The interferon‐related pathway of Reactome enrichment in CD11b + cells of the non‐lymphocytic layer of the spleen in mice treated with KK2DP7. c‐d) Gene set enrichment analysis (GSEA) was conducted on genes related to the NOD‐ and RIG‐I‐like receptor signal pathway in CD11b + cells treated with KK2DP7 compared to those treated with PBS. The top core genes and their interactions in the NOD, RIG‐I, and interferon signaling pathways were displayed. Furthermore, a heatmap of transcription factor expression in RNA and ATAC sequences was generated. h) A genome browser track demonstrating differentially accessible regions associated with differential transcription factors. i) An analysis of GO functional networks regulated by differential transcription factors. j) An analysis of KEGG signaling pathway networks regulated by differential transcription factors.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Protein-Protein interactions, Expressing, Generated, Functional Assay

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: The induction of training immunity by KK2DP7 is dependent on the involvement of TLR2‐IRF7 signaling. a) Display of p‐values and expression levels of the top core genes in differential signaling pathways. b) Transcription factor regulatory gene network analysis diagram. c) Mechanistic map of KK2DP7's regulation of trained immunity. d) Up‐regulation of TLR2 expression levels in CD45 + CD11b + cells in the non‐lymphocyte layer of mouse spleen after KK2DP7 training (n=3). e‐g) The validation of TLR2, IRF7, and p‐IRF7 expression before and after immune training with KK2DP7 in wild‐type mice, TLR2 knockout mice, and IRF7 knockout mice (n=3). h‐j) The CD11b + cells in the splenic nonlymphocytic layer of TLR2 knockout mice and IRF7 knockout mice did not exhibit notable alterations in the secretion of immune‐associated cytokines following KK2DP7 induction. This was in stark contrast to the observed changes in wild‐type mice (n=3). k) Flowchart of inoculation and KK2DP7 administration in knockout mice. l) The average tumor growth curve of mice in different groups (n=6). m) The survival rates of mice in different groups. n) Individual tumor growth curves from l. o) Flow chart of KK2DP7 combined with anti‐PD‐1 antibody in the treatment of mouse tumors. p) The average tumor growth curve of mice in different groups (n=6). q) The survival rates of mice in different groups. r) Individual tumor growth curves from p. All values presented in this figure are expressed as the mean ± s.d., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. In contrast, a student's t‐test was utilized for two‐group comparisons. Survival curves were obtained using the Kaplan–Meier method and compared by the log‐rank test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Expressing, Protein-Protein interactions, Biomarker Discovery, Knock-Out, Comparison

Journal: Discover Oncology

Article Title: Integrated single-cell and functional analyses define strategies to enhance dendritic cell immunostimulatory capacity

doi: 10.1007/s12672-026-04815-6

Figure Lengend Snippet: Effects of different CD40 agonistic agents on DC maturation and activation. A Viability of mDC following treatment with different CD40 antibodies or untreated. B Proportion of CD83⁺ DCs and representative flow cytometry histograms in different groups, with representative flow cytometry histograms. C IL-12p70 secretion of DC in different groups. D DCs treated as indicated were co-cultured with autologous CD14⁻ cells, followed by ELISpot assays to assess T-cell activation. Quantitative analysis and representative images of IFN-γ⁺ spots are shown. Data are presented as mean ± SD (n = 3). *P < 0.05,***P < 0.001, ****P < 0.0001 vs. Control

Article Snippet: On day 6, the culture medium was replaced with serum-free DC medium containing the synthetic p53 R175H peptide and maturation factors, and cells were incubated at 37 °C, 5% CO2 for 20 h. Three hours prior to harvesting, PD-L1 antibody Atezolizumab (10 μg/mL, Roche) and CD40 antibody Cifurtilimab (1 μg/mL, MCE) were added.

Techniques: Activation Assay, Flow Cytometry, Cell Culture, Enzyme-linked Immunospot, Control

Journal: Discover Oncology

Article Title: Integrated single-cell and functional analyses define strategies to enhance dendritic cell immunostimulatory capacity

doi: 10.1007/s12672-026-04815-6

Figure Lengend Snippet: Combination of anti-PD-L1 and CD40 agonist enhanced DC maturation and function. A Viability of mDCs treated with PD-L1 antibody alone or in combination with CD40 antibody. B Proportion of CD83⁺ DCs and representative flow cytometry histograms in the two groups. C IL-12p70 secretion levels of DCs following different treatments. D DCs were co-cultured with autologous CD14⁻ cells, and T-cell activation was evaluated by ELISpot assay showing IFN-γ⁺ spot quantification and representative images. E DCs, either peptide-pulsed or unpulsed, were treated with the combination of anti-PD-L1 antibody and CD40 agonist or left untreated, and then co-cultured with autologous CD14⁻ T cells. T-cell activation was evaluated by IFN-γ ELISpot assay, with quantified spot numbers and representative images shown. Data are presented as mean ± SD (n = 4). *P< 0.05, **P < 0.01, ****P < 0.0001

Article Snippet: On day 6, the culture medium was replaced with serum-free DC medium containing the synthetic p53 R175H peptide and maturation factors, and cells were incubated at 37 °C, 5% CO2 for 20 h. Three hours prior to harvesting, PD-L1 antibody Atezolizumab (10 μg/mL, Roche) and CD40 antibody Cifurtilimab (1 μg/mL, MCE) were added.

Techniques: Flow Cytometry, Cell Culture, Activation Assay, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: (A) Representative flow cytometric histograms showing the specificity of dimeric CD20 aptamer WB1/1.CD20.1_3S across CD20-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (B) Bar graph quantifying the mean fluorescence intensities of WB1/1.CD20.1_3S binding, highlighting significant specificity (*p < 0.0001). (C; E) Fluorescence histograms for CD19 homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (D; F) Quantification of homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, binding specificity using mean fluorescence intensity. (G) Fluorescence histograms for CD19 heterodimeric aptamer WB15/17.CD19.1_3S, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (H) Quantification of heterodimeric aptamer WB15/17.CD19.1_3S binding specificity using mean fluorescence intensity. (I-L) Confocal microscopy images showing colocalization of bivalent WB1/1.CD20.1_3S (L1-L2: Cy3, RED) with a CD20-specific antibody (J1-J2: APC, GREEN) on Raji cells. DNA random aptamer controls and isotype antibody controls confirmed specificity. The aptamer binds to the cell surface membrane, as shown in zoomed-in views (L1-L2). Panel M-P: Confocal microscopy images of bivalent WB17/17.CD19.1_3S (M1-M2: Cy3, RED) colocalizing with a CD19-specific antibody (N1-N2: APC, GREEN) on Raji cells. The aptamer demonstrates specificity and surface binding with no significant off-target interactions. Zoomed-in views (P1-P2) confirmed aptamer binding to the cell membrane. Scale bars = 7 and 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence. The aptamer and random mean fluorescence values correspond to the mean fluorescence observed in their respective histograms. Bar graphs represent mean ± standard deviation from three independent experiments with statistical significance indicated (****p < 0.0001). Data represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Fluorescence, Binding Assay, Confocal Microscopy, Membrane, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization studies of CD19 Antibody and Bivalent CD19 Aptamers in Ramos and Raji Cells. (A–H) Time-course analysis of CD19 antibody and bivalent CD19 aptamer internalization in Ramos cells (A, C, E, and G: CD21-negative) and Raji cells (B, D, F, and H: CD21-positive). (A-B) demonstrate internalization of APC-CD19 antibody in Ramos (A) and Raji (B) cells. In Ramos cells (A), CD19 antibody shows efficient internalization over 48 hours, while in Raji cells (B), CD21 expression inhibits internalization. (C-D) show internalization of bivalent CD19 aptamer WB17/17.CD19.1_3S in Ramos (C) and Raji (D) cells. WB17/17.CD19.1_3S shows robust internalization in Ramos cells (C), but reduced uptake in Raji cells owing to CD21-mediated blocking. (E-F) illustrate internalization of bivalent CD19 aptamer WB15/17.CD19.1_3S in Ramos (E) and Raji (F) cells. The aptamer demonstrates high internalization efficiency in Ramos cells (E), but not CD21-positive Raji cells (F). Panels G and H: Internalization of bivalent CD19 aptamer WB15/15.CD19.1_3S in Ramos (G) and Raji (H) cells. Similar to other bivalent aptamers, WB15/15.CD19.1_3S internalizes efficiently in CD21-negative Ramos cells (G), but CD21 expression in Raji cells (H) significantly blocks its internalization. Data are expressed as the percentage of internalization calculated as . Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Blocking Assay, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Colocalization of CD19 and CD21 was confirmed by flow cytometry and confocal microscopy, whereas CD20 does not co-localize with CD21. (A) demonstrates flow cytometry histograms showing CD19 (A1: PE-CD19 mAb, Red) and CD21 (A2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (A3) Bi-parametric dot plot confirms colocalization of CD19 and CD21 on the same population of cells. (B) shows bar graph quantifying mean fluorescence intensity of CD19 and CD21, highlighting their robust expression on Raji cells. (C–E) Confocal microscopy images showing CD19 (C: PE-CD19, Red) colocalized with CD21 (D: APC-CD21, Light Blue) on the surface of Raji cells. Arrowheads indicate regions of colocalization (D). Panels F1–F3 illustrate flow cytometry analysis of bivalent CD19 aptamer WB17/17.CD19.1_3S (F1: Pink) binding to Raji cells and its colocalization with CD21 (F2: APC-CD21 mAb, Light Blue). (F3) Dot plot shows overlapping signals, confirming aptamer-CD21 interaction. (G) demonstrates the bar graph of mean fluorescence intensity of bivalent CD19 aptamer (WB17/17.CD19.1_3S) and CD21 antibody. (H–J) present confocal microscopy images showing WB17/17.CD19.1_3S (H: Red) colocalized with CD21 (I: Green) on Raji cells. Arrowheads highlight colocalized regions (J). (K1–K3) Flow cytometry analysis showing CD20 (K1: FITC-CD20 mAb, Green) and CD21 (K2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (K3) Dot plot shows no significant overlap between CD20 and CD21, indicating no colocalization. (L) Bar graph showing fluorescence intensity of CD20 and CD21. (M–O) Confocal microscopy images showing no colocalization between CD20 (M: Red) and CD21 (N: Green) on Raji cells (O). (P1–P3) Flow cytometry analysis of bivalent CD20 aptamer WB1/1.CD20.1_3S (P1: Blue) and CD21 (P2: APC-CD21 mAb, Light Blue). (P3) Dot plot confirms the absence of colocalization on the same population of cells. Panel Q: Bar graph of fluorescence intensity for CD20 aptamer and CD21. (R–T) Confocal microscopy images showing that WB1/1.CD20.1_3S (R: Red) and CD21 (S: Green) do not co-localize on Raji cells (T). Scale bars = 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Data represents mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Flow Cytometry, Confocal Microscopy, Fluorescence, Expressing, Binding Assay, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: The expression of CD19, CD20, and CD21, the binding activity of bivalent CD19 aptamers, and the internalization dynamics of CD19 antibody and bivalent aptamers in OCl-LY7 and HBL-1 (DLBCL) cells. Antibody staining for CD19, CD20, and CD21 expression. (C; G) demonstrate flow cytometry histograms showing the expression of CD19 (C2: Red), CD20 (C1: Green), and CD21 (C3: Light Blue) on OCI-Ly7 (C), whereas HBL-1 (G) cells express CD19 (G2: Red), CD20 (G1: Green), and CD21 (G3: Light Blue). Isotype controls (gray) confirm specific binding. (D; H) Bar graphs showing mean fluorescence intensities, confirming robust expression of CD19 and CD20, but the absence of CD21 expression in both cell lines. Binding assay with bivalent CD19 aptamers (A1–A4, E1–E4). Fluorescence intensity histograms of bivalent CD19 aptamers (WB17/17.CD19.1_3S, WB15/17.CD19.1_3S, and WB15/15.CD19.1_3S) compared to random DNA control in OCI-Ly7 (A1–A4) and HBL-1 (E1–E4) cells. (B; F) illustrate bar graphs of mean fluorescence intensities showing high binding specificity of bivalent aptamers compared to controls. Internalization of CD19 antibody with and without Proteinase K (I, K). Flow cytometry histograms (L1) and bar graphs (L2) showing internalization dynamics of CD19 antibody in OCI-Ly7 (I), whereas flow cytometry histograms (K1) and bar graphs (K2) show internalization dynamics of CD19 antibody in HBL-1 (K) cells. Internalization was analyzed at 0 hour and 24 hours in the presence and absence of Proteinase K. Data reveal a significant reduction in surface fluorescence intensity after Proteinase K treatment, confirming internalization. Internalization of bivalent CD19 aptamers under the same conditions (J, L). Fluorescence intensity histograms (J1-J3, L1-L3) and bar graphs (J4 and L4) show the internalization of bivalent CD19 aptamers (WB17/17.CD19.1_3S (J2, L2), WB15/17.CD19.1_3S (J1, L1), and WB15/15.CD19.1_3S (J3, L3)) in OCI-Ly7 (J) and HBL-1 (L) cells. Internalization is measured at 0 hour and 24 hours with and without Proteinase K, demonstrating the effective uptake of bivalent aptamers. Data are expressed as the percentage of internalization calculated as fluorescence intensity was calculated using the formula. Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Each bar represents mean ± standard deviation from three independent experiments. Data are presented as mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Binding Assay, Activity Assay, Staining, Flow Cytometry, Fluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization Assay of CD19 Antibody and Bivalent CD19 Aptamer in Ramos and OCI-Ly7 Cells Visualized by Confocal Microscopy. Internalization of CD19 Antibody in Ramos Cells (A-D). Confocal images showing surface-bound APC-CD19 antibody (A1-A2: Red) at 0 hour with and without Hoechst nuclear staining (A2: Blue). (B1–B2) demonstrate the isotype control at 0 hour. Panels C1-C2 show the internalization of APC-CD19 after 24 hours (C1-C2: Red) with and without Hoechst nuclear staining (C2: Blue). (E) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in Ramos Cells (F-I). Initial binding of WB17/17.CD19.1_3S (F1-F2: Green) at 0 hours, as shown with Hoechst-stained nuclei (F2: Blue). (H1–H2) demonstrate internalization of WB17/17.CD19.1_3S at 24 hours with and without Hoechst nuclear staining (H2: Blue). (J) shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Internalization of CD19 Antibody in OCI-Ly7 Cells (K-N) Surface-bound APC-CD19 antibody at 0 hours (K1-K2). (M1–M2) illustrate reduced surface-bound fluorescence at 24 hours, consistent with internalization. After 24 hours, CD19 antibody is completely uptaken on OCl-Ly7 cells. (O) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in OCI-Ly7 Cells (P-S). Confocal images showing WB17/17.CD19.1_3S (P1: Green) binding at 0 hours with and without Hoechst nuclear staining (P2: Blue). (R1–R2) highlight internalized WB17/17.CD19.1_3S at 24 hours with Hoechst nuclear staining (R2). Panel T shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Scale Bars: 5 μm. Data represent mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Confocal Microscopy, Staining, Control, Fluorescence, Binding Assay, Standard Deviation