Journal: Cold Spring Harbor Molecular Case Studies

Article Title: Tumor-immune microenvironment revealed by Imaging Mass Cytometry in a metastatic sarcomatoid urothelial carcinoma with a prolonged response to pembrolizumab

doi: 10.1101/mcs.a006151

Figure Lengend Snippet: Full list of antibodies and their properties

Article Snippet: CD20 , Dy161 , L26 , 1:200 , 0.5 , Novus Biologicals , NBP2-80486.

Techniques: Concentration Assay

Journal: Materials Today Bio

Article Title: Sophora flavescens-derived extracellular vesicles loaded with oncolytic vaccinia virus/IR1061 for NIR-II photoacoustic imaging guided multimodal treatment of diffuse large B-cell lymphoma

doi: 10.1016/j.mtbio.2025.102177

Figure Lengend Snippet: SFOVV@IR1061 induces apoptosis and ICD in A20 lymphoma cells. (A) Flow cytometric analysis of apoptosis in A20 cells treated with PBS, SFNPs, OVV, IR1061 + laser, OVV@IR1061+laser, or SFOVV@IR1061 + laser (1064 nm, 5 min, 0.8 W/cm 2 ) for 12 h. Cells were stained with Annexin V-FITC and propidium iodide (PI). (B) Representative CLSM images of live/dead cell staining using Calcein-AM (green, live cells) and PI (red, dead cells) under the same treatment conditions. (C) Quantification of the dead/live cell ratio from CLSM images shown in (B). Data are presented as mean ± SD (n = 3). (D) Western blot analysis of apoptosis-related proteins including PARP, and Caspase-3 in A20 cells treated as indicated. β-actin was used as a loading control: 1. PBS; 2. SFNPs; 3. OVV; 4. IR1061+laser; 5. OVV@IR1061+laser; 6. SFOVV@IR1061+laser. (E) Western blot analysis of ICD-related markers CRT, HSP70, and HMGB1 following the same treatments: 1. PBS; 2. SFNPs; 3. OVV; 4. IR1061+laser; 5.OVV@IR1061 +laser; 6. SFOVV@IR1061+laser. (F) Flow cytometric analysis of macrophage polarization markers CD86 (left) and CD206 (right) in RAW264.7 cells. (G) Quantification of fluorescence intensity for CD86 and CD206. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To analyze M1/M2 macrophage polarization, cells were incubated with FITC Anti-Mouse CD86 Antibody (Elabscience, China) to detect M1 polarization and APC Anti-Mouse CD206 Antibody (Elabscience, China) to detect M2 polarization, according to the manufacturer's instructions.

Techniques: Staining, Western Blot, Control, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Association of CCL21 + CAFs with B-cell recruitment and TLS maturation in penile squamous cell carcinoma

doi: 10.1007/s00262-026-04348-5

Figure Lengend Snippet: Evaluation of TLS maturation and prognostic analysis of PSCC patients. A UMAP plot of the cell types identified in PSCC through spatial transcriptomic sequencing, depicting 7 distinct cell populations. B Dot plot displaying the expression of cell type-specific markers. The dot size represents the proportion of cells expressing the marker, while the colour intensity indicates the mean expression level. C Regions with AUCell scores for mTLS genes ( BCL6 , AICDA , CD38 , ICOS , CXCR5 , CXCL13 , and CD21 ) above the median were defined as mTLSs, whereas those below the median were classified as iTLSs. D Spatial visualization of AUCell scores for mTLS genes, where warmer colours indicate higher scores. E Spatial distribution of iTLSs (green) and mTLSs (orange) across different regions of PSCC tissues. F Representative images H&E staining and immunofluorescence staining for CD20 (green) and CD21 (red) in iTLSs and mTLSs. G GO enrichment analysis of upregulated genes in mTLS. Darker colours denote more significant enrichment of the pathway, and larger dots represent a greater number of genes involved. H Kaplan–Meier survival curves showing that patients with mTLSs experienced significantly longer overall survival than those with iTLSs, as determined using ssGSEA ( P < 0.05)

Article Snippet: The following antibodies were used: CD20 (Proteintech, 60,271–1-Ig, 1:500), CD21 (Proteintech, 28,206–1-AP, 1:250), ACTA2/α-SMA (Sigma, SAB5700835, 1:300), and CCL21 (Abcam, ab9851, 1:300).

Techniques: Sequencing, Expressing, Marker, Staining, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Association of CCL21 + CAFs with B-cell recruitment and TLS maturation in penile squamous cell carcinoma

doi: 10.1007/s00262-026-04348-5

Figure Lengend Snippet: Characterization of CCL21 + CAF subtypes and their association with TLS maturation in PSCC. A UMAP plot of CAF subtypes in PSCC derived from spatial transcriptomics, including CCL21 + CAFs (green), COL16A1 + CAFs (blue), MYH11 + CAFs (orange), and XBP1 + CAFs (red). B Dot plot of key markers identified across CAF subtypes. The dot size represents the proportion of cells expressing the marker, and colour intensity indicates the mean expression level. C Radar plot showing the distribution of CCL21 + CAF proportions across the iTLS-core, mTLS-core, iTLS-edge, and mTLS-edge regions. D Bar chart illustrating the spatial distances between different CAF subtypes and mTLS structures; ***, P < 0.001. E In situ visualization of TLS regions: green areas indicate the iTLS-core, blue circles indicate the iTLS-edge, orange areas represent the mTLS-core, and yellow circles denote the mTLS-edge. F Spatial localization of CCL21 + CAFs in PSCC tissues showing their association with distinct TLS regions. G Heatmap overlay of the CCL21 + CAF spatial density and TLS distribution, with red indicating a higher CCL21 + CAF abundance. H Scatter plots of the correlation between the CCL21 + CAF proportion and B-cell (left panel) and naive T-cell (right panel) numbers in the iTLS/mTLS regions. I Representative images of mIHC of TLS regions in PSCC showing the spatial distribution and colocalization of CD20 (purple), ACTA2/α-SMA (green), and CCL21 (yellow). Scale bar = 150 μm. ( J ) Patients with high CCL21 + CAF signature scores (n = 29) experienced significantly longer overall survival than those with low scores (n = 26)

Article Snippet: The following antibodies were used: CD20 (Proteintech, 60,271–1-Ig, 1:500), CD21 (Proteintech, 28,206–1-AP, 1:250), ACTA2/α-SMA (Sigma, SAB5700835, 1:300), and CCL21 (Abcam, ab9851, 1:300).

Techniques: Derivative Assay, Spatial Transcriptomics, Expressing, Marker, In Situ

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: A) This diagram depicts the dual‐targeting strategy of biHSNPs in facilitating tumor cell elimination. Each biHSNP features two functional arms: one specifically binds to a tumor‐associated antigen on lymphoma cells (CD19/CD20), while the other engages and activates immune effector cells (CD3/CD16). By bridging tumor cells and immune cells, this approach enhances immune‐mediated tumor cell lysis, leveraging the immune system's inherent ability to identify and eliminate abnormal cells for precise and efficient cancer cell killing. B) Schematic illustration of the T cell activation and tumor cell death mechanism mediated by biHSNPs.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Functional Assay, Lysis, Activation Assay

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Schematic illustration of the chemical synthesis and characterization of antibody‐conjugated hollow silica nanoparticles. A) Synthetic pathway of PEGylated hollow silica nanoparticles. B) Fourier transform‐infrared spectroscopy (FT‐IR) characterization of HSNP@PEG (2). C) Proton nuclear magnetic resonance ( 1 H NMR) characterization of HSNP@PEG. D) DLS and TEM images of HSNP@PEG. E) DLS and TEM images of HSNP@PEG@NH 2 (3). F) Fluorescence spectrum of HSNP@PEG@NH 2 @FITC (9.62 µ m ), confirming successful FITC conjugation. G) DLS and TEM images of HSNP@PEG@NH 2 @FITC (4). H–K) DLS and TEM results for monospecific nanoparticles (HSNP αCD3 , HSNP αCD16 , HSNP αCD19 , HSNP αCD20 ). L–O) DLS and TEM results for bispecific nanoparticles (HSNP αCD3 + αCD19 , HSNP αCD3 + αCD20 , HSNP αCD16 + αCD19 , HSNP αCD16 + αCD20 ). All scale bars in the TEM images represent 100 nm. HSNP αCD3 , HSNP αCD16 , HSNP αCD19 and HSNP αCD20 : Monospecific HSNPs conjugated with a single type of antibody (anti‐CD3, anti‐CD16, anti‐CD19, or anti‐CD20, respectively). HSNP αCD3 + αCD19 , HSNP αCD3 + αCD20 , HSNP αCD16 + αCD19 and HSNP αCD16 + αCD20 : Bispecific HSNPs conjugated with two different types of antibodies (e.g., anti‐CD3 and anti‐CD19). Antibody types are indicated using lowercase Greek letters (e.g., αCD3).

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Fourier Transform Infrared Spectroscopy, Spectroscopy, Nuclear Magnetic Resonance, Fluorescence, Conjugation Assay

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Binding specificity and cell–cell linkage induced by biHSNPs. Target cells were incubated with FITC‐labeled, antibody‐coated HSNPs at 4 °C for 30 min, followed by flow cytometry analysis to confirm binding specificity. A–C) Depict cell–cell linkage induced by biHSNPs: A) Schematic illustration of cell‐cell complex formation mediated by biHSNPs. B) Confocal microscopy images showing stable junctional complexes between immune effector cells and tumor cells facilitated by HSNP αCD3 + αCD19 and HSNP αCD3 + αCD20 . C) Flow cytometry analysis of cell–cell complexes between pre‐stained Jurkat (green, CellTracker Green CMFDA) and Raji cells (red, CellTracker Red CMTPX), demonstrating effective linkage by biHSNPs. D) Verification of biHSNPs binding to CD3 + cells using PBMCs as the CD3 + cell line. E) Verification of biHSNPs binding to CD19 + /CD20 + cells using Raji cells as the target. F) Verification of biHSNPs binding to CD16 + cells using PBMCs as the CD16 + cell line. G) Verification of biHSNPs binding to CD19 + /CD20 + cells using Raji cells as the target.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry, Confocal Microscopy, Staining

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: A) Cytotoxicity and cytokine release assays of biHSNPs in luciferase‐transfected Raji cells. B) In vitro cytotoxicity of HSNP@PEG, HSNP αCD3 , HSNP αCD19 , and bispecific HSNP αCD3 + αCD19 . C) In vitro cytotoxicity of HSNP@PEG, HSNP αCD3 , HSNP αCD20 , and bispecific HSNP αCD3 + αCD20 . D,E) Cytokine secretion levels (IFN‐γ, IL‐2, and TNF‐α) for HSNP αCD3 , HSNP αCD19 , and HSNP αCD3 + αCD19 ; and HSNP αCD20 and HSNP αCD3 + αCD20 . F) In vitro cytotoxicity of HSNP@PEG, HSNP αCD16 , HSNP αCD19 , and bispecific HSNP αCD16 + αCD19 . G) In vitro cytotoxicity of HSNP@PEG, HSNP αCD16 , HSNP αCD20 , and bispecific HSNP αCD16 + αCD20 . H,I) Cytokine secretion levels (IFN‐γ, IL‐2, and TNF‐α) for HSNP αCD16 , HSNP αCD19 , HSNP αCD16 + αCD19 , HSNP αCD20 , and HSNP αCD16 + αCD20 . J) Cytotoxicity of combined bispecific nanoparticle treatments: HSNP αCD3 + αCD19 with HSNP αCD16 + αCD19 , and HSNP αCD3 + αCD20 with HSNP αCD16 + αCD20 . K) In vitro cytotoxicity of HSNP αCD3 + αCD19 combined with HSNP αCD16 + αCD19 , and HSNP αCD3 + αCD20 combined with HSNP αCD16 + αCD20 . Data are presented as mean ± SD ( n ≥ 3), Statistical differences in B–I) were analyzed by One‐way ANOVA and K) were analyzed by Student's t‐test and the statistical significance is indicated as ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Luciferase, Transfection, In Vitro

Journal: Oncology letters

Article Title: Immunohistochemical profile and prognostic significance in primary central nervous system lymphoma: Analysis of 89 cases.

doi: 10.3892/ol.2017.6893

Figure Lengend Snippet: Figure 1. Immunohistochemical labeling. (A and B) H&E staining. In tumor cells with the diffuse distribution, the size of nuclei were 2 times greater than of normal lymphocytes. (A) Magnification, x200. (B) Magnification, x400. (C) CD20 cell membrane staining performed using the EnVision method. Magnification, x200. (D) CD10 cell membrane staining performed using the EnVision method. Magnification, x200. (E) BCL‑6, nuclei staining, EnVision method, x100. (F) MUM‑1 nuclei staining performed using the EnVision method. Magnification, x200. (G) BCL‑2 cytoplasmic staining performed using the EnVision method. Magnification, x200. (H) Ki‑67 nuclei staining performed using the EnVision method. Magnification, x200. H&E, hematoxylin and eosin; CD, cluster of differentiation; BCL, B cell lymphoma; MUM‑1, multiple myeloma‑1.

Article Snippet: Monoclonal antibodies against CD20 (UM800002, 1:100 dilution), CD10 (UM870127, 1:600 dilution), BCL-6 (TA804186, 1:150 dilution), MUM1 (TA327705; 1:100‐1:500 dilution), BCL-2 (UM870117, 1:500 dilution), Ki-67 (TA352729, 1:100), CD138 (TA327619, 1:25-1:200) were used and all purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Immunohistochemical staining, Labeling, Staining, Membrane

Journal: bioRxiv

Article Title: CD20 + T cells are associated with inflammatory responses in experimental arthritis

doi: 10.1101/2021.10.12.464081

Figure Lengend Snippet: ( A ) Flow cytometry dot-plots depicting approach used to gate on CD3 + , CD4 + , and CD8 + T cells. ( B and C ) Dot-plots showing representative Naïve, Non-Sick, and CIA mice, depicting CD20 expression by CD4 + and CD8 + T cells. ( D and E ) Percentages of CD3 + CD4 + CD20 + and CD3 + CD8 + CD20 + T cells in Naïve (n=7), Non-Sick (n=5) and CIA (n=7) mice. *p<0.05, **p<0.01. ns, not significant.

Article Snippet: Samples were stained with a rabbit anti-mouse CD20 antibody (1:100) (Cell Signaling, catalog number: 98708S) and a biotinylated hamster anti-mouse CD3 antibody (STEMCELL, catalog number: 60015BT.1; 1:100) at 4°C overnight.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: CD20 + T cells are associated with inflammatory responses in experimental arthritis

doi: 10.1101/2021.10.12.464081

Figure Lengend Snippet: The frequency of GM-CSF ( A ), TNF-α ( B ), IL-17 ( C ), and IFN-γ ( D ) producing CD3 + CD4 + CD20 + T cells in Naïve, Non-Sick, and CIA mice. ( E-H ) Percentages of GM-CSF, TNF-α, IL-17 and IFN-γ expression by CD3 + CD8 + CD20 + T cells in Naïve, Non-Sick and CIA mice. Naïve, n=7; Non-Sick, n=5; CIA, n=7. *p<0.05, **p<0.01; ***p<0.001. ns, not significant.

Article Snippet: Samples were stained with a rabbit anti-mouse CD20 antibody (1:100) (Cell Signaling, catalog number: 98708S) and a biotinylated hamster anti-mouse CD3 antibody (STEMCELL, catalog number: 60015BT.1; 1:100) at 4°C overnight.

Techniques: Expressing

Journal: bioRxiv

Article Title: CD20 + T cells are associated with inflammatory responses in experimental arthritis

doi: 10.1101/2021.10.12.464081

Figure Lengend Snippet: Lymph nodes ( A ) and joint tissue ( B ) from mice undergoing CIA were collected and fixed in formaldehyde. Sections were stained with specific anti-CD3 (Magenta) and anti-CD20 (Cyan) antibodies. DAPI (grey) was used to stain nucleic acid as counterstaining. Scale bars: 100 μm. Superimposed yellow elements indicate as follows; [dotted lines]: bone limits, [asterisks]: synovial CD20 + CD3 + cells. Images were acquired using a confocal microscope.

Article Snippet: Samples were stained with a rabbit anti-mouse CD20 antibody (1:100) (Cell Signaling, catalog number: 98708S) and a biotinylated hamster anti-mouse CD3 antibody (STEMCELL, catalog number: 60015BT.1; 1:100) at 4°C overnight.

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: CD20 + T cells are associated with inflammatory responses in experimental arthritis

doi: 10.1101/2021.10.12.464081

Figure Lengend Snippet: ( A ) Flow cytometry dot-plots depicting CXCR5 and PD-1 expression by total CD4 + T cells. ( B, C ) Percentages of CXCR5 + PD-1 + and CXCR5 - PD-1 + T cells by total CD3 + CD4 + T cells. ( D ) Flow cytometry dot-plots depicting CXCR5 and PD-1 expression within CD3 + CD4 + CD20 + T cells in representative Naïve, Non-Sick, and CIA mice. ( E, F ) Percentages of CXCR5 + PD-1 + and CXCR5 - PD-1 + T cells among CD3 + CD4 + CD20 + T cells, respectively. ( G ) Flow cytometry dotplots depicting expression of CXCR5 and PD-1 within CD3 + CD4 + CD20 - T cells. ( H, I ) Percentages of CXCR5 + PD-1 + and CXCR5 - PD-1 + T cells among CD3 + CD4 + CD20-T cells, respectively. ( J ) Flow cytometry dot-plots depicting CXCR5 and PD-1 expression by total CD3 + CD8 + T cells. ( K, L ) Percentages of CXCR5 + PD-1 + and CXCR5 - PD-1 + T cells within CD3 + CD8 + T cells. ( M ) Flow cytometry dot-plots depicting expression of CXCR5 and PD-1 within CD3 + CD8 + CD20 + T cells. ( N, O ) Percentages of CXCR5 + PD-1 + and CXCR5 - PD-1 + T cells among CD3 + CD8 + CD20 + T cells, respectively. ( P ) Flow cytometry dot-plots depicting expression of CXCR5 and PD-1 within CD3 + CD8 + CD20 - T cells. ( Q, R ) Percentages of CXCR5 + PD-1 + and CXCR5 - PD-1 + T cells within CD3 + CD8 + CD20 - T cells. Naïve, n=7; Non-Sick, n=5; and CIA mice, n=7. *p<0.05, **p<0.01. ns, not significant.

Article Snippet: Samples were stained with a rabbit anti-mouse CD20 antibody (1:100) (Cell Signaling, catalog number: 98708S) and a biotinylated hamster anti-mouse CD3 antibody (STEMCELL, catalog number: 60015BT.1; 1:100) at 4°C overnight.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: CD20 + T cells are associated with inflammatory responses in experimental arthritis

doi: 10.1101/2021.10.12.464081

Figure Lengend Snippet: Flow cytometry dataset files from single, live CD4 + or CD8 + T cells from non-arthritic and arthritic joints were used as input for the opt-SNE embedding using Cytobank. ( A ) CD20, CXCR5, and PD-1 expression by CD4 + T cells of non-arthritic and arthritic mice are depicted. ( B-D ) Mean fluorescence intensity (MFI) of CD20, CXCR5, and PD-1 and summary data are presented. ( E ) CD20, CXCR5, and PD-1 expression by CD8 + T cells of non-arthritic and arthritic mice are shown. ( F-H ) Mean fluorescence intensity (MFI) of CD20, CXCR5, and PD-1 in CD8 + T cells and summary data are presented. Non-arthritic mice, n=4; Arthritic mice; n=4.

Article Snippet: Samples were stained with a rabbit anti-mouse CD20 antibody (1:100) (Cell Signaling, catalog number: 98708S) and a biotinylated hamster anti-mouse CD3 antibody (STEMCELL, catalog number: 60015BT.1; 1:100) at 4°C overnight.

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Dysregulated lymphocyte localization in idiopathic multicentric Castleman disease

doi: 10.64898/2025.12.12.693846

Figure Lengend Snippet: (A) UMAP of B cell sub-cluster highlighting GC B cells. (B) Quantification and comparison of total GC B cells in Infl. CTRL (n=4) and iMCD-TAFRO (n=4). (C) IF images of GCs using B cell marker CD20 and (D) quantification of cells within the GC. p-value is as indicated. (E) UMAP projection of T/NK cell sub-cluster indicating Tfh-like cells in Infl CTRL and iMCD-TAFRO and (F) quantification. (G) Within the Tfh-like cell cluster, differential gene expression comparing iMCD-TAFRO to Infl. CTRL indicates a down-regulation of GC-Tfh genes (grey) and up-regulation of anti-migration-associated genes (red) in iMCD-TAFRO Tfh-like cells. (H) GC-Tfh enrichment scores were applied to the Tfh-like subcluster in both groups. (I) Quantification of the average GC-Tfh activity scores per patient. (J) Representative IF images of GC-Tfh cells that express CD3, PD1, and BCL6 within GCs and (K) quantification. Dots in bar graphs represent individual patient samples. *p<0.05.

Article Snippet: Histology sections (5 um) of FFPE tissue from each sample was cut fresh and stained with antibodies against CD3 (Novus # NBP2-54392AF532) and CD20 (Novus # NBP2-47840AF594) and nuclear dye Sytox13.

Techniques: Comparison, Marker, Gene Expression, Migration, Activity Assay