Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD21 , REA940 , 50 , 130-115-609 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD21 , REA940 , 50 , 130-115-609 , FITC , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: Hemophagocytic lymphohistiocytosis presenting with annular erythema multiforme-like eruptions in a patient with angioimmunoblastic T cell lymphoma: A case report

doi: 10.3892/etm.2018.6420

Figure Lengend Snippet: Histological and immunohistochemical staining findings of the patient. A biopsy sample taken from the left cervical lymph node revealed that normal structure had disappeared, and small to medium-sized atypical lymphoid cells had infiltrated the lymph node region. (A) H&E staining; original magnification, ×10. (B) H&E staining; original magnification, ×40. (C-G) On immunohistochemical staining, the lymphoid cells were diffusely positive for CD2, CD3, CD5, CD7 and CD10 (H&E staining; original magnification, ×40). (H) CD20, (I) paired-box domain 5 and (J) telomerase B staining were weak positive (H&E staining; original magnification, ×40). (K) CD21 staining suggested follicular dendritic cells net damage (H&E staining; original magnification, ×40). (L) Proliferative index with Ki-67 stain was high (H&E staining; original magnification, ×40). H&E, hematoxylin and eosin; CD, cluster of differentiation.

Article Snippet: The antibodies utilized included: with CD2 (cat. no. UMAB86), CD3 (cat. no. UM570048), CD5 (cat. no. UM570009), CD7 (cat. no. TA506337), CD10 (cat. no. TA590055), CD20 (cat. no. UM800065), CD21 (cat. no. TA327627) and Ki-67 (TA801577) all diluted at 1:100. paired-box domain 5 (cat. no. TA801884) and telomerase B (cat. no. TA301588) were diluted at 1:50 (all antibodies were obtained from OriGene Technologies, Inc.) and hematoxylin staining as previously described ( 17 ).

Techniques: Immunohistochemical staining, Staining

Journal: European journal of immunology

Article Title: Essential role of antigen-presenting cell-derived BAFF for antibody responses.

doi: 10.1002/eji.200636791

Figure Lengend Snippet: Figure 1. Phenotypic characterisation and purity of sorted PBMC. (A) Expression of SWC1 on APC. CD4/CD172a dot plot defining four subsets of APC and their SWC1 expression (histograms). (B) SWC1 expression on T and NK cells. (C) SWC1/ sIg expression on PBMC, and SWC1/CD21 expression on sIg+

Article Snippet: Phenotypic analysis For phenotypic analysis, cells were stained using murine mAb against SWC1, CD172a, CD6, CD3 (mAb 8E6; VMRD), CD8 (mAb 11-295-33; kindly provided by Prof. Dr. A. Saalm ller, University of Veterinary Medicine Vienna), CD21 (mAb BB6-11C9.6; SouthernBiotech), TCR1 d chain (VMRD), goat mAb against human BAFF (R&D Systems) and polyclonal affinity-purified goat anti-swine antibody (Jackson Immuno Research).

Techniques: Expressing

Journal: Oncotarget

Article Title: Inflammaging increases susceptibility to cigarette smoke-induced COPD

doi: 10.18632/oncotarget.4027

Figure Lengend Snippet: A. Representative micrographs of HE-stained lung tissue sections from young and old FA and CS-exposed mice showing the presence of iBALT in aged animals after CS exposure; scale bar 100 μm. B. Total volume of iBALT per basal membrane was determined via quantitative morphological assessment. C. Lung tissue mRNA expression of CXCL13 was measured by qPCR. D. Representative micrographs of immunohistochemical stainings for CD3 (T lymphocytes), B220 (B lymphocytes) and CD21 (FDCs) in lung sections from CS-exposed young and old mice; scale bar 100 μm. E. Flow cytometry analysis of whole lung for CD4 + T lymphocyte infiltration reveals increases in Th2 and Th17 cells after CS exposure in aged compared to young mice; Th1 cells were defined by expression of CD4 and IFNγ, Th2 cells by expression of CD4 and IL-4, Th17 cells by expression of CD4 and IL-17A, and regulatory T cells by expression of CD4, CD25 and Foxp3. F. Lung tissue mRNA expression of Th signature cytokines IFNγ and IL-13 and transcription factors Gata3, Rorγt and Foxp3 was measured by qPCR. Data were combined from 2 independent experiments with n = 8 and are given as mean values ± SD; one-way ANOVA following Bonferroni post test with * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After overnight incubation with primary antibodies against MMP12 (Abcam, Cambridge, UK), CD45R/B220 (BD Pharmingen), CD21 (Novus Biologicals, Littleton, CO), CD3 (Sigma Aldrich), p16 (Santa Cruz, CA) or SIRT1 (Millipore, Schwalbach, Germany) tissue slides were incubated with an alkaline phosphatase-labeled secondary antibody (Biocare Medical).

Techniques: Staining, Membrane, Expressing, Immunohistochemical staining, Flow Cytometry

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Synergistic activation by MB‐CD40L, BCR, and TLR‐9 signaling for improved B‐cell expansion and differentiation. (a) Representative flow cytometry plot of CD27 versus CD38 for days 0–13 cells (gated on IgD‐ B‐cells from day‐4 onwards) for the activation of naïve B‐cells by MB‐CD40L with or without the B‐cell receptor signal by anti‐human IgM/G/A (H + L) antibody plus co‐receptor signal by anti‐human CD21 antibody (referred to as BCR) and/or toll‐like receptor signal by the TLR‐9 agonist CpG ODN 2006 in the presence of BAFF and the interleukins Il‐2, ‐4, ‐10 and ‐21. Bar graphs of (b) fold expansion and the yield of (c) memory B‐cells and (d) ASCs on day‐13 for each group. Data were acquired each day as duplicates for each group and represented as mean + standard deviation. Statistical analysis was conducted by applying ordinary one‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns p > 0.05).

Article Snippet: For the activation of the B‐cell receptor, a combination of goat anti‐human IgA + IgG + IgM (H + L) antibody (109006064; Jackson Immuno Research Laboratories, USA) was added to the culture at 6 μg/mL concentration, together with a monoclonal mouse anti‐human CD21 antibody (MAB4909; R&D Systems, USA) at 1 μg/mL.

Techniques: Activation Assay, Flow Cytometry, Standard Deviation

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Switching interleukins and CD40L dosage for the terminal differentiation of naïve B‐cells for effective generation of functional ASCs. (a) Representative flow cytometry plot of CD27 versus CD38 gated on IgD‐ B‐cells on day‐10 and day‐13. High‐purity naïve B‐cells were activated with MB‐CD40L, BAFF, anti‐BCR/CD21 antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for initial 10 days. On day‐10, CD40L dosage was either maintained (MB‐CD40L) or reduced by 10‐fold (MB‐CD40L low ). For each CD40L dosage, three different conditions were tested up to day‐13: All factors or all factors except IL‐21 or all factors except anti‐BCR antibodies and IL‐4. (b), (c) Bar graphs comparing the yield percentage of memory B‐cells (IgD‐CD38‐/lo + CD27) and ASCs (IgD‐CD38++CD27+) on day‐13 from indicated conditions. Data were acquired as duplicates and presented as mean + standard deviation. (d) Number of ASCs generated by day‐13 from the culture of 10,000 naïve B‐cells (day‐0). Either MB‐CD40L or Sol Multimer CD40L was employed for the entirety of the culture period. Other indicated culture conditions were varied between day‐10 and day‐13. Data were acquired as triplicates and presented as mean + standard deviation. (e), (f) The concentrations of antibodies of different isotypes, IgM and IgG, secreted by ASCs generated by activation of high‐purity naïve B‐cells using the indicated method of CD40L presentation in the presence of BAFF, anti‐BCR antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for the initial 10 days. On day‐10, cells were reseeded at 10,000 cells per well, CD40L dosage was reduced by 10‐fold, and all soluble factors except anti‐BCR antibodies and IL‐4 were employed. ELISA was performed using supernatant samples harvested on day‐12. Data were acquired as triplicates and presented as mean + standard deviation. (b)−(f) Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001; ns p > 0.05).

Article Snippet: For the activation of the B‐cell receptor, a combination of goat anti‐human IgA + IgG + IgM (H + L) antibody (109006064; Jackson Immuno Research Laboratories, USA) was added to the culture at 6 μg/mL concentration, together with a monoclonal mouse anti‐human CD21 antibody (MAB4909; R&D Systems, USA) at 1 μg/mL.

Techniques: Functional Assay, Flow Cytometry, Standard Deviation, Generated, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

doi: 10.1371/journal.pone.0171171

Figure Lengend Snippet: CD21 + purified B cell populations were cultured in the presence or absence of CD40L (1.0 ug/ml), gated on live cells, and then analyzed for changes in the median fluorescence intensity (MFI) of SLA-DR (A) and CD80 (B). Cells were analyzed prior to culture and at 24 h intervals for a period of 7 days. One representative experiment of two performed is displayed. Data shown are mean ± SEM. Statistical differences of * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 are indicated.

Article Snippet: The following antibodies were used: unlabeled goat anti-pig IgM and IgG polyclonal antibodies (Ab) (Bethyl), mouse anti-porcine IgM monoclonal antibody (mAb) (AbD Serotec K52 1C3), hamster anti-mouse CD80 PE-Cy7 mAb, mouse anti-pig SLA Class II DR mAb (AbD Serotec 2E9/13), horseradish peroxidase (HRP)-conjugated goat anti-pig IgM and IgG polyclonal Ab (Bethyl); phycoerythrin (PE)-labeled mouse anti-porcine CD21 mAb (Acris Antibodies BB6-11C9.6); FITC-goat anti-pig IgG polyclonal Ab (Bethyl); and eFluor 780-Fixable Viability Dye (eBioscience).

Techniques: Purification, Cell Culture, Fluorescence

Journal: PLoS ONE

Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

doi: 10.1371/journal.pone.0171171

Figure Lengend Snippet: Quiescent or CD40L activated CD21 + cells were stimulated with IL-4 or IL-21 at 50 ng/ml. (A) CD21 + cells were stained with CFSE and analyzed by flow cytometry at day 7 of culture for proliferation. (B) Proliferating percentages for each treatment condition. (C) Total cell numbers after 7 days of culture. (D) Cell culture viability dynamics. (E) IgM production from activated and stimulated cells, quantified with a total IgM ELISA of culture supernatant. (F) Cell viability evaluated by fixable viability dye staining and flow cytometry at day 7. One representative experiment of three with three different animals is displayed. Data are mean ± SEM. Statistical differences of *p < 0.05, ** p < 0.01, ***p < 0.001 were determined using ANOVA to compare group means followed by t-tests to calculate significance.

Article Snippet: The following antibodies were used: unlabeled goat anti-pig IgM and IgG polyclonal antibodies (Ab) (Bethyl), mouse anti-porcine IgM monoclonal antibody (mAb) (AbD Serotec K52 1C3), hamster anti-mouse CD80 PE-Cy7 mAb, mouse anti-pig SLA Class II DR mAb (AbD Serotec 2E9/13), horseradish peroxidase (HRP)-conjugated goat anti-pig IgM and IgG polyclonal Ab (Bethyl); phycoerythrin (PE)-labeled mouse anti-porcine CD21 mAb (Acris Antibodies BB6-11C9.6); FITC-goat anti-pig IgG polyclonal Ab (Bethyl); and eFluor 780-Fixable Viability Dye (eBioscience).

Techniques: Staining, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

doi: 10.1371/journal.pone.0171171

Figure Lengend Snippet: CD21+ purified B cells were treated with cytokines as described and cultured in technical triplicates. Culture supernatants and cells were collected and analyzed at day 7 for number of total cells via hemocytometer (A) and flow cytometry with fixable viability dye (B). Total secreted IgM (C) and IgG (D) were determined by ELISA as described in Materials and Methods. Results are representative of three independent experiments with three individual animals. Data are mean ± SEM. Statistical differences of *p < 0.05, ** p < 0.01, ***p < 0.001 were determined using ANOVA to compare group means followed by t-tests to calculate significance.

Article Snippet: The following antibodies were used: unlabeled goat anti-pig IgM and IgG polyclonal antibodies (Ab) (Bethyl), mouse anti-porcine IgM monoclonal antibody (mAb) (AbD Serotec K52 1C3), hamster anti-mouse CD80 PE-Cy7 mAb, mouse anti-pig SLA Class II DR mAb (AbD Serotec 2E9/13), horseradish peroxidase (HRP)-conjugated goat anti-pig IgM and IgG polyclonal Ab (Bethyl); phycoerythrin (PE)-labeled mouse anti-porcine CD21 mAb (Acris Antibodies BB6-11C9.6); FITC-goat anti-pig IgG polyclonal Ab (Bethyl); and eFluor 780-Fixable Viability Dye (eBioscience).

Techniques: Purification, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

doi: 10.1371/journal.pone.0171171

Figure Lengend Snippet: CD21 + B cells were cultured in triplicate in the indicated cytokines to determine the effects on IgM and IgG ASC differentiation at 4 days of culture (A). Cells were evaluated at 24 h intervals up to 7 days for live IC IgM + (B) and IgG + (C) cell percentages via flow cytometry, using the gating strategy and results for day 4 of culture displayed in panel A. On day 7, culture supernatants and cells were collected and analyzed for secreted IgM (D) and IgG (E) by ELISA. Total cells (F) were counted using a hemocytometer. Fixable viability dye and flow cytometry were used to determine cellular viability and then multiplied by total cell counts to calculate total live cell counts (G). Data (mean ± SEM) shown are from one of three equivalent experiments with three different animals. Statistical differences of *p < 0.05, ** p < 0.01, ***p < 0.001 were determined using ANOVA to compare group means followed by t-tests to calculate significance.

Article Snippet: The following antibodies were used: unlabeled goat anti-pig IgM and IgG polyclonal antibodies (Ab) (Bethyl), mouse anti-porcine IgM monoclonal antibody (mAb) (AbD Serotec K52 1C3), hamster anti-mouse CD80 PE-Cy7 mAb, mouse anti-pig SLA Class II DR mAb (AbD Serotec 2E9/13), horseradish peroxidase (HRP)-conjugated goat anti-pig IgM and IgG polyclonal Ab (Bethyl); phycoerythrin (PE)-labeled mouse anti-porcine CD21 mAb (Acris Antibodies BB6-11C9.6); FITC-goat anti-pig IgG polyclonal Ab (Bethyl); and eFluor 780-Fixable Viability Dye (eBioscience).

Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

doi: 10.1371/journal.pone.0171171

Figure Lengend Snippet: CD21 + cells were cultured in triplicate in the indicated cytokines. On day 4 of culture, cells were incubated with unlabeled anti-IgM, then stained for flow cytometry with a fixable viability dye, CD21, Annexin V, and then fixed and permeabilized and stained for IC IgM (A). Annexin V bright (B) and dim (C) populations were gated to show the live and dead distribution of the two populations. One representative experiment of two performed with two different animals is displayed.

Article Snippet: The following antibodies were used: unlabeled goat anti-pig IgM and IgG polyclonal antibodies (Ab) (Bethyl), mouse anti-porcine IgM monoclonal antibody (mAb) (AbD Serotec K52 1C3), hamster anti-mouse CD80 PE-Cy7 mAb, mouse anti-pig SLA Class II DR mAb (AbD Serotec 2E9/13), horseradish peroxidase (HRP)-conjugated goat anti-pig IgM and IgG polyclonal Ab (Bethyl); phycoerythrin (PE)-labeled mouse anti-porcine CD21 mAb (Acris Antibodies BB6-11C9.6); FITC-goat anti-pig IgG polyclonal Ab (Bethyl); and eFluor 780-Fixable Viability Dye (eBioscience).

Techniques: Cell Culture, Incubation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

doi: 10.1371/journal.pone.0171171

Figure Lengend Snippet: Splenocytes from four PRRSV immune and two naïve animals were enriched for CD21 + B cells. (A) Cells were labeled with CFSE and then cultured in the presence of CD40L, APRIL, and BAFF with or without IL-21. At day 3 of culture, 1/3 of cells were harvested and evaluated for IC IgG + and CFSE divisions via flow cytometry. (B) PRRSV nsp7 (100,000 live cells/well) and IgG + ELISPOT (10,000 live cells/well) were performed on 1/3 of cultured cells. Following normalization of plated cell number, the proportion of nsp7-specific spots to IgG + spots was calculated and is displayed in (C). The remaining IL-21 treated cells were cultured for a total of 14 days. Supernatants were harvested and evaluated with a limiting dilution nsp7 ELISA. Individual titers are displayed above corresponding columns in (C). Data are representative of two independent experiments with the same animals.

Article Snippet: The following antibodies were used: unlabeled goat anti-pig IgM and IgG polyclonal antibodies (Ab) (Bethyl), mouse anti-porcine IgM monoclonal antibody (mAb) (AbD Serotec K52 1C3), hamster anti-mouse CD80 PE-Cy7 mAb, mouse anti-pig SLA Class II DR mAb (AbD Serotec 2E9/13), horseradish peroxidase (HRP)-conjugated goat anti-pig IgM and IgG polyclonal Ab (Bethyl); phycoerythrin (PE)-labeled mouse anti-porcine CD21 mAb (Acris Antibodies BB6-11C9.6); FITC-goat anti-pig IgG polyclonal Ab (Bethyl); and eFluor 780-Fixable Viability Dye (eBioscience).

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