Journal: PLoS ONE

Article Title: Uptake and Presentation of Myelin Basic Protein by Normal Human B Cells

doi: 10.1371/journal.pone.0113388

Figure Lengend Snippet: PBMCs from healthy donors were incubated with or without 30 µg/ml MBP in medium containing normal autologous serum (30% v/v), or in pure medium. The resulting deposition of C3 and C1q on B cells was measured by flow cytometry after 5 min incubation (N = 3). Representative histogram plots show A) C3-deposition, and B) C1q-deposition on B cells. C) The binding of MBP was assessed using biotinylated MBP as probe and subsequent staining with streptavidin-PE. Blockade of CR1 or CR2 was achieved by pre-incubation of PBMCs with mAb3D9 and polyclonal sheep anti-human CR2 respectively. Monoclonal anti-glycophorin (GP)-A was used as negative control. D) Mean fluorescence intensity (MFI) values of 5–6 experiments are shown; background values (of samples with no MBP added) have been subtracted. Bars and error bars represent means and SEM. **p<0.01, ***p<0.001.

Article Snippet: Murine anti-human CR1 IgG1 antibody (mAb3D9) was kindly donated by Dr John O'Shea (Frederick Cancer Research and Development Center, Frederick, MD, USA), and polyclonal sheep anti-human CR2 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Flow Cytometry, Binding Assay, Staining, Negative Control, Fluorescence

Journal: PLoS ONE

Article Title: Uptake and Presentation of Myelin Basic Protein by Normal Human B Cells

doi: 10.1371/journal.pone.0113388

Figure Lengend Snippet: PBMCs from healthy HLA-DR15+ donors were incubated for 18 h with MBP in media containing normal serum. Cells were stained with FITC anti-CD19 and biotinylated MK16, followed by streptavidin-PE. (A) The binding of MK16 at different serum concentrations is shown as mean fluorescence (MFI) values normalised to that of 10% serum, (N = 4). B) Before addition of serum (30% v/v), different concentrations of the complement inhibitory compound sodium polyanethole sulphonate (SPS) were added. MFI values are shown, normalised to samples without SPS, (N = 6). (C) The PBMCs were pre-incubated with the anti-CR1 mAb3D9 or polyclonal sheep anti-human CR2, or both, before addition of serum (30% v/v) and MBP. Anti-glycophorin (GP)-A was used as negative control. Data are shown as means±SEM, (N = 4–6). **p<0.01.

Article Snippet: Murine anti-human CR1 IgG1 antibody (mAb3D9) was kindly donated by Dr John O'Shea (Frederick Cancer Research and Development Center, Frederick, MD, USA), and polyclonal sheep anti-human CR2 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Staining, Binding Assay, Fluorescence, Negative Control

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: iScience

Article Title: Salmonella infection induces the reorganization of follicular dendritic cell networks concomitant with the failure to generate germinal centers

doi: 10.1016/j.isci.2023.106310

Figure Lengend Snippet:

Article Snippet: Biotin Recombinant monoclonal anti-CD21/35 (REA800) , Miltenyi Biotec , Cat# 130-111-650; RRID: AB_2656312.

Techniques: Purification, Recombinant, Virus, Saline, Plasmid Preparation, Microscopy, Reverse Transcription, Software

Journal: OncoTargets and Therapy

Article Title: Correlation of C-X-C chemokine receptor 2 upregulation with poor prognosis and recurrence in human glioma

doi: 10.2147/ott.s91626

Figure Lengend Snippet: Figure 1 High level of CXCR2 expression shown in cases of high-grade gliomas. Notes: (A) Representative sections for CXCR2 immunohistochemistry (IHC, SP ×400). (a) Normal nontumorous tissue, (b) WHO low-grade glioma, (c) WHO high-grade glioma, (d) negative control for immunostaining. Positive CXCR2 staining = brown; cell nuclei = blue; the arrows show representative results of staining. (B) The population of cells with different levels of CXCR2 expression in glioma and control brain tissue. Overall, the level of CXCR2 expression was significantly higher in WHO III–IV gliomas tissues than in WHO I–II glioma tissues and the control brain tissues according to IHC results. (C) Western blotting of CXCR2 protein level in gliomas and normal tissue. The upper panel is a representative result of Western blotting. CXCR2 protein expression was calculated by normalizing CXCR2 intensity to GAPDH intensity, and data were compared to the normal tissue, represented as 1. Data are expressed as mean ± SD; *P,0.05 versus normal tissue; #P,0.05 between different grades. Abbreviations: CXCR2, C-X-C chemokine receptor 2; WHO, World Health Organization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.

Article Snippet: Western blotting was performed according to standard protocols using the following antibodies: goat anti-glyceraldehyde 3-phosphate dehydrogenase polyclonal antibody (1:800; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-human CXCR2 monoclonal antibody (1:500); donkey anti-goat IRDye (1:10,000; Rockland, Limerick, PA, USA), and donkey anti-mouse IRDye (1:10,000; Rockland).

Techniques: Expressing, Immunohistochemistry, Negative Control, Immunostaining, Staining, Control, Western Blot, Standard Deviation

Journal: OncoTargets and Therapy

Article Title: Correlation of C-X-C chemokine receptor 2 upregulation with poor prognosis and recurrence in human glioma

doi: 10.2147/ott.s91626

Figure Lengend Snippet: Figure 2 CXCR2 inhibitor SB225002 could reduce cell migration. Notes: (A) Representative images for wound healing assay. U251 cells treated with DMSO or with SB225002 (400 nM) at the different time points of 0 hour, 12 hours, 24 hours, 36 hours, and 48 hours, respectively (bar 100 µm). (B) Statistical results of the wound healing assay. Images were analyzed using ImageJ analysis software and data presented as average length of cell-free void ± SD (*P,0.05). Abbreviations: CXCR2, C-X-C chemokine receptor 2; DMSO, dimethyl sulfoxide; SD, standard deviation; h, hour.

Article Snippet: Western blotting was performed according to standard protocols using the following antibodies: goat anti-glyceraldehyde 3-phosphate dehydrogenase polyclonal antibody (1:800; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-human CXCR2 monoclonal antibody (1:500); donkey anti-goat IRDye (1:10,000; Rockland, Limerick, PA, USA), and donkey anti-mouse IRDye (1:10,000; Rockland).

Techniques: Migration, Wound Healing Assay, Software, Standard Deviation

Journal: Breast Cancer (Tokyo, Japan)

Article Title: B-cell populations are expanded in breast cancer patients compared with healthy controls

doi: 10.1007/s12282-017-0824-6

Figure Lengend Snippet: Identifying B-cell subsets using surface marker profiles and fluorescence-activated cell sorting (FACS). a Schematic showing cellular subsets during B-cell differentiation and the cell surface markers that are expressed on the corresponding B-cell subsets. Firstly, immature B cells enter the bloodstream from the bone marrow before eventually reaching the central arterioles and then the marginal zone (MZ) of the spleen. The immature B cells then become transitional B cells, and are classified as either T1, T2, or T3. T1 B cells express CXCR5 on their surface and migrate from the MZ region to the B region of the spleen by interacting with CXCL13 secreted by follicular dendritic cells, and mature into T2 and then T3 B cells before leaving the spleen as mature, naive B cells. Naive B cells have yet to recognize antigen and comprise approximately 60% of peripheral blood B cells. These cells are characterized as CD27 − and recognize antigens through IgM- and IgD-type receptors. When naive B cells migrate to the lymph nodes, where they recognize antigens, they then differentiate at the germinal center to become memory B cells, expressing cell surface IgG, IgA, and IgE, or, occasionally, IgM. Naive B cells may also be activated to differentiate directly into antibody-producing plasma cells. Finally, when memory B cells circulating in the peripheral blood encounter an antigen for a second time, they may become a plasma cell and rapidly produce large numbers of high affinity antibodies. The common white blood cell antigen CD45 and the B-cell marker CD19 are expressed at all stages from immature B cell to plasmablast. Transitional B cells are reported to express CD5 [26]. While CD24 is expressed at the more immature T1/T2 stages, CD21 expression increases as the cells mature towards the T2/T3 stage. Mature naive B cells express high levels of IgD, memory B cells express CD27, and antibody-producing plasma cells express CD27 and CD38. b Representative FACS data depicting normal PBMCs taken from a 49-year-old woman. The arrow denotes the fraction that has been expanded. (i) Firstly, forward scatter (FSC) and side scatter (SSC) were measured, and the lymphocyte gate was defined. (ii) CD45 + cells were selected from the lymphocyte fraction. (iii) Cells highly positive for both IgD and CD45 were defined as Naive B cells. (iv) CD45 + cells were selected and then CD19 (horizontal axis) and CD5 (vertical axis) positivity was assessed. (v) CD45 + /CD19 + /CD5 − cells were selected and then the CD38 and CD27 positivity of this subset, shown on the horizontal and vertical axes, respectively, was assessed. CD45 + /CD19 + /CD5 − /CD38 + cells were defined as plasmablast cells and CD45 + /CD19 + /CD5 − /CD27 + cells were defined as memory B cells. (vi) CD45 + /CD19 + /CD5 + cells were selected and then the CD38 and CD27 positivity of this subset, shown on the vertical and horizontal axes, respectively, was assessed. (vii) CD45 + /CD19 + /CD5 + /CD38 + /CD27 − cells were selected and levels of CD24 and CD21 positivity, shown on the horizontal and vertical axes, respectively, was assessed. The CD24 + and CD21 + cells were defined as T1 and T3 transitional B cells, respectively

Article Snippet: APC-conjugated anti-human CD21 antibody (clone FAB4909A) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Marker, Fluorescence, FACS, Cell Differentiation, Expressing, Clinical Proteomics