fitc conjugated rat anti cd169  (Bio-Rad)

Journal: International Journal of Molecular Sciences

Article Title: Heterogeneity of Phenotypic and Functional Changes to Porcine Monocyte-Derived Macrophages Triggered by Diverse Polarizing Factors In Vitro

doi: 10.3390/ijms24054671

Figure Lengend Snippet: Effect of diverse polarizing factors on porcine moMΦ surface marker expressions (mean of fluorescence intensity). Porcine moMΦ were left untreated, or they were stimulated with diverse polarizing factors: IFN-γ + LPS (both at 100 ng/mL), IL-4 (20 ng/mL), IL-10 (20 ng/mL), TGF-β (20 ng/mL), or dexamethasone (20 ng/mL). Then, 24 h post-stimulation, flow cytometry was employed to determine the expression of several surface markers: MHC I, MHC II DR, CD14, CD16, CD163, and CD169. Mean fluorescence intensity (MFI) of positive cells was evaluated, and MFI data are expressed as fold change relative to the un-activated condition (moMΦ). Data from three independent experiments utilizing different blood donors are presented. Data are displayed as box-and-whisker plots, showing the median and interquartile range (boxes) and minimum and maximum values (whiskers). Values of treated macrophages were compared to the untreated control (moMΦ), using an unpaired t -test of a Mann–Whitney test. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: Cells were first stained with Zombie Aqua viability dye (BioLegend, San Diego, CA, USA) (30 min, room temperature), then they were washed with PBS supplemented with 0.5% bovine serum albumin (BSA), and subsequently stained with several murine monoclonal antibodies (mAbs): anti-porcine CD16-PE (clone G7, Thermo Scientific Pierce, Rockford, IL, USA), anti-human CD14-PerCP-Cy5.5 (clone Tuk4, Miltenyi Biotec, Bergisch Gladbach, Germany) [ ], CD163-PE (clone 2A10/11, Bio-Rad Antibodies, Kidlington, UK), CD169-FITC (clone 3B11/11, Bio-Rad Antibodies), anti-pig MHC I (clone JM1E3, Bio-Rad Antibodies), and anti-pig MHC II DR (clone 2E9/13, Bio-Rad Antibodies) ( ).

Techniques: Marker, Fluorescence, Flow Cytometry, Expressing, Whisker Assay, Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CK2β-regulated signaling controls B cell differentiation and function

doi: 10.3389/fimmu.2022.959138

Figure Lengend Snippet: Phenotypic characterization of splenic B cell subsets in CK2β CTRL and CK2β κO mice. (A) Scatter plots summarizing the percentage of T1 (B220 + CD19 + CD21 low CD23 - IgM hi ), T2 (B220 + CD19 + CD21 low CD23 + IgM hi ) and T3 (B220 + AA4.1 + IgM low CD23 + ) B cells in the spleen of CK2β CTRL and CK2β κO mice, with each symbol representing a mouse. Statistical significance was determined by Student’s t test (*p < 0.05). (B) Scatter plots showing the percentages of MZP (CD21 hi CD23 + ) B cells in the spleen of CK2β CTRL and CK2β κO mice. Data are represented as mean± SD. Statistical significance was determined by Student’s t test (***p < 0.001). (C) Right, Scatter plots representing the percentage of splenic B220 + CD19 + IgM +/- IgD + and B220 + CD19 + IgM + IgD - B cells of CK2β CTRL and CK2β κO mice, with each symbol representing a mouse. Data are shown as mean ± SD. Statistical significance was determined by Student’s t test (**p < 0.01). Left, one representative dot plot is shown for each genotype. (D) (Right) Scatter plots summarizing the percentage of MZ (B220 + CD19 + CD21 hi CD23 - ) and Fo (B220 + CD19 + CD21 dim/- CD23 + ) B cells in the spleen of CK2β CTRL and CK2β κO mice. Statistical significance was determined by Student’s t test (***p < 0.001; ****p < 0.0001). Left, one dot plot is presented for each genotype, numbers in gates indicate the percentages of Fo and MZ B cells. (E) Histogram summarizing the absolute number of Fo and MZ B cells in CK2β CTRL and CK2β κO mice. Data are shown as mean ± SD (n=7). Statistical significance was determined by Student’s t test (*p < 0.05; ***p < 0.001). (F) Spleen sections from CK2β CTRL and CK2β κO mice were stained with H&E. Bar, 500μm upper panels; 100μm lower panels. Image acquisition was performed using the Leica DMD108 Digital Microimaging Device and Software (Leica Microsystems, Germany). Data show results from one representative mouse out of 3. Arrows indicate the MZ. (G) Ratio between MZ and lymphoid follicle areas in the spleen of CK2β CTRL and CK2β κO mice was calculated using the Leica DMD108 Digital Microimaging Device and Software (2 mice per genotype; 2 independent experiments; +/+= 32 follicles; fl/fl= 54 follicles). Data are shown as mean ± SD. Statistical significance was determined by Student’s t test (*p < 0.05). (H) CD169 (green), IgD (blue) and IgM (red) expression in spleen sections from CK2β CTRL and CK2β κO mice was analyzed by IF. One representative mouse out of 3 per genotype is shown; 3 independent experiments. Bar, 50μm. Images were acquired with Zeiss LSM 700 confocal microscope and ZEN software. Pictures were acquired using 10x/0.3 dry and 20x/0.8 dry objectives at room temperature and merged in three-color images with ImageJ software. (I) Quantification of the percentage of T1, MZ and Fo B cells that incorporated BrdU after continuous administration for 6 days in CK2β CTRL (n=5) and CK2β κO (n=4) mice. Three independent experiments. MZ, marginal zone.

Article Snippet: Abs used: FITC anti-CD169 (MOMA1; AbD Serotec), PE anti-IgM (R6-60.2, BD), V450 anti-IgD (R6-60.2; BD).

Techniques: Staining, Software, Expressing, Microscopy

Journal: Frontiers in Immunology

Article Title: CK2β-regulated signaling controls B cell differentiation and function

doi: 10.3389/fimmu.2022.959138

Figure Lengend Snippet: Activation of the NOTCH2 pathway determines an expansion of the MZ. (A) Splenic CD19 + B cells analyzed for the expression of Hes1 and Dtx1 by qRT-PCR. The expression is corrected for Gapdh levels and normalized to CK2β CTRL B cells. Data are shown as mean ± SD (n=4, three independent experiments). Statistical significance was determined by Student’s t test (***p < 0.001). (B) Splenic B cells analyzed for the expression of Notch2 by qRT-PCR. The expression is corrected for Gapdh levels and normalized to CK2β CTRL B cells. Data are shown as mean ± SD (n=5, three independent experiments). (C) Top, NOTCH2 expression in CK2β CTRL and CK2β KO splenic B lymphocytes was determined by WB (whole cell lysates, one representative experiment out of three). Bottom, Mean relative density of three experiments relative to CK2β CTRL B cells. Data are shown as mean ± SD. Statistical significance was determined by Student’s t test (*p < 0.05). In (A-C) the B cell fraction was purified using EasySep™ Mouse B Cell isolation kit (Stemcell) and the purity was ≥97%. (D) Flow cytometry analysis of NOTCH2 expression through intracytoplasmic staining. Top, NOTCH2 MFI in CK2β CTRL and CK2β KO mice, Bottom, graphs summarizing the percentage of NOTCH2 positive cells in the gate of MZ and Fo B cells shown as mean ± SD (two independent experiments). Statistical significance was determined by Student’s t test (*p < 0.05). (E) MZ B cells (CD21 hi CD23 - ) from CK2β CTRL and CK2β κO mice were analyzed by flow cytometry after IgG or α-NRR2 administration. Top, representative dot plots indicating the MZ B cell gate. Bottom, Histograms summarizing the data of two independent experiments shown as mean ± SD. Statistical significance was determined by Student’s t test (*p < 0.05). (F) IF images of spleen sections showing CD169 (green), IgD (blue) and IgM (red) expression in IgG and α-NRR2 treated mice. Bar, 50μm. One representative mouse out of two per group is shown. Images were acquired with Zeiss LSM 700 confocal microscope and analyzed with ZEN software. Pictures were acquired using objectives 10x/0.3 dry and 20x/0.8 dry at room temperature and merged in three-color images with ImageJ software.

Article Snippet: Abs used: FITC anti-CD169 (MOMA1; AbD Serotec), PE anti-IgM (R6-60.2, BD), V450 anti-IgD (R6-60.2; BD).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Purification, Cell Isolation, Flow Cytometry, Staining, Microscopy, Software

Journal: PLoS ONE

Article Title: Subcapsular Sinus Macrophage Fragmentation and CD169 + Bleb Acquisition by Closely Associated IL-17-Committed Innate-Like Lymphocytes

doi: 10.1371/journal.pone.0038258

Figure Lengend Snippet: (A) Flow cytometric detection of IL-7Rα expression on CD169 + CD11c lo cells from digested lymph nodes. (B) Immunofluorescence microscopy of a lymph node section stained with anti-CD169 and anti-IL-7Rα monoclonal antibodies. Enlargements (bottom, far right) show examples of IL-7Rα + cells (white arrows) closely associated with CD169 + SSMs. FO, follicle; T, T zone. (C) Flow cytometric analysis showing CD169 + CD11c lo cells contain a population of IL-7Rα hi CCR6 + cells. All data in A–C are representative of at least three independent experiments. (D) Flow cytometric analysis of digested lymph node cells from a control and TCRβδ-deficient mouse. IL-7Rα hi CCR6 + cells gated from total cells express CD169 on a fraction of both CD3e + and CD3e − cells. Data are representative of at least three independent experiments (control mice) and one experiment in which a TCRβδ-deficient mouse was analyzed.

Article Snippet: FITC-conjugated anti-CD169 ( clone 3D6.122) was purchased from AbD Serotec.

Techniques: Expressing, Immunofluorescence, Microscopy, Staining

Journal: PLoS ONE

Article Title: Subcapsular Sinus Macrophage Fragmentation and CD169 + Bleb Acquisition by Closely Associated IL-17-Committed Innate-Like Lymphocytes

doi: 10.1371/journal.pone.0038258

Figure Lengend Snippet: (A) Flow cytometric detection of CD169 on digested lymph node cells stained with two anti-CD169 monoclonal antibodies, Ser4 and 3D6. The top panel is pre-gated on IL-7Rα hi CCR6 + cells; the bottom panel shows total cells. Data are representative of three independent experiments. (B) Expression of CD169 on total cells from digested lymph nodes from CD169-DTR mice treated with saline or DT 3 or 4 days prior to analysis. Data are representative of two independent experiments. (C) Siglec1 mRNA quantification by RT-PCR in sorted CD169 + and CD169 – IL-7Rα hi CCR6 + cells, CD169 + CD11c lo F4/80 – cells, and CD169 + CD11c lo F4/80 + cells. Data are plotted relative to HPRT. (D) Immunofluorescence microscopy of lymph nodes stained with anti-CD169 and anti-CD11b monoclonal antibodies from a Siglec1 –/– bone marrow chimeric mouse (top panel) or control non-chimeric mouse (bottom). Scale bar = 50 µm. Data are representative of one experiment. (E) Flow cytometric detection of CD169 staining on CD45.1 + Siglec1 +/+ radiation-resistant (blue) compared to donor bone-marrow-derived CD45.2 + Siglec1 –/– (red) IL-7Rα hi CCR6 + cells. CD169 + staining on IL-7Rα hi CCR6 + cells from a non-chimeric control animal are represented in black. Data are representative of two experiments. (F) Flow cytometric detection of CD169 and CD11b on IL-7Rα hi CCR6 + cells from digested lymph nodes. Data are representative of two experiments. (G) CD169 + IL-7Rα hi CCR6 + B220 − cells from digested lymph nodes were sorted and fixed to a slide for immunofluorescence microscopy. Data are representative of one experiment.

Article Snippet: FITC-conjugated anti-CD169 ( clone 3D6.122) was purchased from AbD Serotec.

Techniques: Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Microscopy, Derivative Assay

Journal: PLoS ONE

Article Title: Subcapsular Sinus Macrophage Fragmentation and CD169 + Bleb Acquisition by Closely Associated IL-17-Committed Innate-Like Lymphocytes

doi: 10.1371/journal.pone.0038258

Figure Lengend Snippet: (A) Gating scheme to identify CD169 + IL-7Rα hi CCR6 + cells analyzed on an ImageStreamX imaging flow cytometer (Amnis Corp). The area of CD169 staining on images of the gated cells was quantified using a Threshold mask on the upper 60% of the pixel intensities (right panel). (B) Representative images of cells with small, medium, and large areas of CD169 area based on the histogram in (A). Channels for CD169 (green), IL-17Rα (red), and brightfield are shown. Graph on right shows the frequency of cells falling in each gate (n = 2 mice, lines indicate means). (C) Gating scheme to identify CD169 + CD11c + cells analyzed on an ImageStreamX imaging flow cytometer. The area of CD169 staining on images of the gated cells was quantified as described in (A). (D) Representative images of cells with small, medium, and large areas of CD169 area based on the histogram in (C). Channels for CD169 (green) IL-17Rα (red), and brightfield are shown. Graph on right shows the frequency of cells falling in each gate (n = 2 mice, lines indicate means). Data are representative of one experiment with two mice. In a second experiment with two mice, CD169 + blebs were visualized on CD169 + CCR6 + TCRγδ + cells.

Article Snippet: FITC-conjugated anti-CD169 ( clone 3D6.122) was purchased from AbD Serotec.

Techniques: Imaging, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Subcapsular Sinus Macrophage Fragmentation and CD169 + Bleb Acquisition by Closely Associated IL-17-Committed Innate-Like Lymphocytes

doi: 10.1371/journal.pone.0038258

Figure Lengend Snippet: (A) Flow cytometric detection of CD169 on digested lymph node cells gated on IL-7Rα hi CCR6 + cells, NK1.1 + DX5 + CD3ε − cells, CD3e + cells, and B220 + cells. (B) Flow cytometric detection of CD169 on IL-7Rα hi CCR6 + cells from digested (top panel) or non-digested (bottom panel) lymph nodes. All data are representative of at least two independent experiments.

Article Snippet: FITC-conjugated anti-CD169 ( clone 3D6.122) was purchased from AbD Serotec.

Techniques:

Journal: PLoS ONE

Article Title: Subcapsular Sinus Macrophage Fragmentation and CD169 + Bleb Acquisition by Closely Associated IL-17-Committed Innate-Like Lymphocytes

doi: 10.1371/journal.pone.0038258

Figure Lengend Snippet: (A) CD44, CD62L and CXCR6 expression on digested lymph node cells from control or Cxcr6 GFP/+ mice, gated on IL-7Rα hi CCR6 + cells. (B) IL-17A staining of digested lymph node cells, stimulated with PMA/I for 2 h, gated on gated on IL-7Rα hi CCR6 + cells. (C) αβT and γδT staining on digested lymph node cells, gated on IL-7Rα hi CCR6 + cells; CD169 staining of αβT + , γδT + and TCR − IL-7Rα hi CCR6 + cells gated as indicated in the left panel. (D) Flow cytometric detection of IL-7Rα hi CCR6 + cells that bind CD1d-tetramers; far right panel shows CD169 staining on CD1d-tetramer + IL-7Rα hi CCR6 + cells. (E) CD169, CD4, and CD8 staining on digested lymph node cells, gated on IL-7Rα hi CCR6 + cells. (F) Immunofluorescence microscopy of a lymph node from a wild-type or a CD4-deficient mouse, stained with anti-CD169 and anti-CD4 monoclonal antibodies. FO, follicle; T, T zone. Scale bar = 50 µm. All data are representative of at least two independent experiments.

Article Snippet: FITC-conjugated anti-CD169 ( clone 3D6.122) was purchased from AbD Serotec.

Techniques: Expressing, Staining, Immunofluorescence, Microscopy