Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Serum IgM from C57BL/6J, muMT, bumble, and A/J mice was measured by ELISA. Serum was harvested from mice either naïve, chronically infected with type III CEP strain, or on D5 post-secondary infection with type I GT1 T. gondii strain. PerC transfer (+) refers to mice adoptively transferred 5×10 6 total PerC cells as a day 2 neonate. Each dot represents the results from an individual mouse, and plotted is the average +/-SD of the O.D. obtained at 450nm; *P<0.05, unpaired two-tailed t-test. B) Bumble reconstitution of the peritoneal B-1 compartment after neonatal PerC adoptive transfer. Representative FACS plots of peritoneal B-2 cells (B220 high CD19+) and B-1 (B220 int-neg CD19+) cells from bumble mice with or without PerC adoptive transfer. Shown are mice on day 20 of primary infection with the type III CEP strain. C) B cell deficient muMT mice (n=3), muMT given WT PerC adoptive transfers as 2-day neonates then allowed to reconstitute for 6-7 weeks into adulthood (n=2), and muMT given B cell enriched splenocytes (n=3) 1 day prior to infection with the type III CEP strain were assessed for survival. D) muMT reconstitution of B-2 cell compartment, WT and muMT with B cell enriched splenocytes (EasySep™ Mouse Pan-B Cell Isolation Kit, cat# 19844) adoptively transferred 24 hrs earlier E) muMT reconstitution of peritoneal B cell compartment after neonatal adoptive transfer. Representative FACS plots of peritoneal B-2 cells (B220high CD19+) and B-1 B cells (B220int-neg CD19+) from WT, and muMT mice or muMT mice with neonatal PerC adoptive transfer. For D and E, uninfected mice are 6-8 weeks of age and numbers indicate the percent of cells that fall within the depicted gate.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Adoptive Transfer Assay, Cell Isolation

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Serum obtained from A/J and C57BL/6J mice chronically infected (CEP) or at D5 of secondary infection (GT1) were used to probe GT1 parasite lysate separated by SDS-PAGE, western blots were detected with anti-mouse IgG. “Total Lane Signal” is the signal obtained from the entire lane of C57BL/6J compared to that of A/J (=1); western blots were developed in tandem and analyzed by Image J. Results are from 6 individual experiments; ** P < 0.01, * P < 0.05; unpaired two-tailed t-tests. B) Gating strategies for identifying memory B cells. Memory B cells are identified as CD19+ B220+ CD23+ CD21 mid CD73+. Conventional memory B cells are FCRL5-CD80-while atypical memory B cells are identified as FCRL5+, CD80+. Representative FACS plots of memory compartments in A/J and C57BL/6J mice on day 5 of secondary infection with the type I GT1 strain are shown. The frequency of class-switched (IgM-IgD-) memory cells at the indicated infection states were analyzed. Each dot represents the results from an individual mouse and the cumulative averages +SD from 2 experiments are plotted. N=3-6 mice per infection state. Significance was assessed with an unpaired two-tailed t-test; *** P<0.0001, ** P<0.01.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Infection, SDS Page, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Gating strategies for identifying B-1 (CD19+ B220 int-neg ) and B-2 (CD19+ B220 hi ) B cells. The legend applies to panels C-H. B) Representative FACS plots of the CD11b+ peritoneal B-1 B cell compartment in A/J and C57BL/6J (B6) mice at naïve, chronic (Chr), and 5 days (D5) post-secondary infection with the GT1 strain. Numbers indicate the percent of cells that fall within the indicated gate. Representative histograms of IgM surface expression and percent of cells that fall within the IgM lo gate of CD5-B-1b cells from A/J (red) and C57BL/6J (blue) at the indicated time points. C) Frequencies of splenic CD43+ B-1a (CD5+) or B-1b (CD5-) that are IgM+IgD- or IgM-IgD-in A/J and C57BL/6J mice at the indicated infection states. D) Representative FACS plots of splenic CD19+ B220 int-neg B cells stained for CD43 and CD5 in A/J and C57BL/6J mice at D5 of secondary infection. Representative CD138 expression on CD5-CD43+ B-1b cells. E) Frequencies of CD43+ splenic B-1a and B-1b cells from A/J and C57BL/6J mice that express BAFFR+, TACI+, or CD138+ at the indicated infection states. F) Heatmap depicting the relative expression of all Ighg transcripts from the indicated B cell population, mouse strain and infection state. G) Representative FACS plots of intracellular IgG (H+L) of B-1b cells, and H) frequency of both peritoneal and splenic B-1a and B-1b cells of A/J and C57BL/6J mice at day 5 of secondary infection. For C, E and H, the cumulative average +SD from 2-4 experiments is plotted and each dot represents the result from an individual mouse; P values calculated by 2-way ANOVA with Tukey correction; **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Infection, Expressing, Staining

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: Transcriptomic analysis of peritoneal B-1a (CD19+ B220 int-neg CD11b+ CD5+), B-1b (CD19+ B220 int-neg CD11b+ CD5-) and B-2 (CD19+ B220 hi CD11b-CD5-) B cells from A/J and C57BL/6J mice was performed using 3’-Tag RNA sequencing. A) Genes that were differentially upregulated in B-1b cells on day 5 of secondary infection compared to naïve mice in the A/J genetic background. P values of differentially expressed genes were calculated using the Benjamini-Hochberg adjustment for false discovery rate, and only those genes that survived significance were included in the heatmap. For comparison, all B-1 compartments in A/J mice are shown for this gene set. A/J B-1 Pathway and GO term enrichment was assessed on the genes presented in the heatmap in A. P values for enrichment analysis were adjusted with the Holm-Bonferroni correction. B) A cluster of genes found to be differentially induced in B-1b cells in A/J compared to C57BL/6J mice on day 5 of secondary infection are plotted as a heat map. C) Gene set enrichment analysis of the rank-ordered list of differentially expressed genes between A/J and C57BL/6J B-1b cells at D5 of secondary infection. Gene set depicted was in the top 10 gene sets ranked by false discovery rate (FDR) after investigating MSigDB’s C7: immunologic signatures collection. Enrichment score is the degree of overrepresentation of a gene set at the top or bottom of a ranked list. NES is the enrichment score after normalizing for gene set size.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: RNA Sequencing Assay, Infection

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Nfkbid expression in CPM (Counts per million) of 3’-Tag RNA-seq reads of the indicated B cell populations obtained from A/J and C57BL/6J mice that were either naïve, chronically infected, or on D5 of secondary infection with the type I GT1 strain. B) Enriched B cells from the PerC and spleen were stimulated with LPS for 2 hrs and Nfkbid transcripts were quantified by qPCR; ** P< 0.01, paired t-test. C) Representative histograms display the detection of parasite-specific IgG1 bound to formaldehyde fixed GFP+ type I GT1 parasites; diluted serums (10 3 ) from C57BL/6J, Nfkbid +/-(C57BL/6J x bumble F1), and A/J mice chronically infected with the type III CEP strain were assayed. Anti-mouse IgG1 background staining in the absence of serum is shown. D) As in C, but quantification of the parasite-specific IgG1 antibody isotype binding over a range of serum concentrations is shown. Plotted is the cumulative average +/-SD of the MFI from 2-3 separate experiments (C57BL/6J n=8; Nfkbid +/-n=7; A/J n=8); significance was assessed by unpaired t-tests and Holm-Sidak corrections comparing A/J vs C57BL/6J (*) or Nfkbid+/- vs C57BL/6J (#); **** P<0.0001, # P<0.05. IgG1 staining was not significantly different between A/J and Nfkbid +/-serums. E) Representative FACS plots of the dump-CD19+ CD138+ plasmablast populations within A/J, C57BL/6J and Nfkbid +/-mice at day 5 of secondary infection with type I GT1 parasites. F) Frequency of B220-CD138+ plasmablasts of total live dump-CD19+ cells. Plotted is the cumulative average +/-SD of 2 separate experiments (n=5 per mouse strain); significance was assessed by unpaired t-tests; * P< 0.05.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Expressing, RNA Sequencing Assay, Infection, Staining, Binding Assay

Journal: Frontiers in Immunology

Article Title: Plasmodium curtails autoimmune nephritis via lasting bone marrow alterations, independent of hemozoin accumulation

doi: 10.3389/fimmu.2023.1192819

Figure Lengend Snippet: BM leukocyte populations long-term after Plasmodium infection. Study of BM leukocytes at 2-, 3- and 4-months post-infection with P. yoelii 17XNL (Py , black), P. berghei ANKA Δlap ( Pb Δlap, blue) and P. berghei ANKA Δpm4-Δbp2 ( Pb Δpm4-Δbp2, green). (A) Percentage of general leukocyte populations out of CD45+ cells: myeloid cells (CD11b + ), B cells (B220 + ), T cells (TCRβ + NK1.1 - ) and NK cells (TCRβ-NK1.1 + ). (B) Frequency out of single cells acquired by flow cytometry: neutrophils (CD11b + Ly6C int Ly6G + ), inflammatory monocytes (CD11b + Ly6C hi Ly6G - ), monocytes (CD11b + Ly6C + Ly6G - ), MHC-II + CD38 + myeloid population, CD4/CD8 activated-memory (a/m) T cells (CD44 + CD62L - ), DCs (CD11c hi MHC-II + ), plasmacytoid DCs (pDCs, PDCA-1 + B220 + ), eosinophils (CD11b + SiglecF + SSC hi ) and hematopoietic precursors (Lin - c-Kit + ). Results show representative data (3 and 5 mice per group) of two independent experiments. Data are represented as mean ± SD. One-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n.s; non-significant results are not indicated.

Article Snippet: The anti-mouse antibodies used for flow cytometry were the following: B220-APC-Cy7 (clone RA3-6B2, Biolegend), CD4-PE (clone GK1.5, Biolegend), CD8a-APC (clone 53-6.7, Biolegend), CD11b-FITC NS BV421 (clone M1/70, BD Biosciences), CD11c-PE and PE-Cy7 (clone N418, Biolegend), CD38-APC (clone 90, Biolegend), CD44-PE-Cy7 (clone IM7, Biolegend), CD45.2-APC (clone 104, Biolegend), CD62L-BV421 (clone MEL-14, Biolegend), c-Kit/CD117-BV421 and PerCP-Cy5.5 (clone 2B8, Biolegend), Ly6-C-APC-Cy7 (clone HK1.4, Biolegend), Ly6-G-PE-Cy7 (clone 1A8, BD Biosciences), MHC-II I-A/I-E-Alexa Fluor 488 (clone M5/114.15.2, Biolegend), NK-1.1-Alexa Fluor 700 (clone PK136, BD Biosciences), PDCA-1/BST2/CD317-PE (clone 129C1, Biolegend), TCRb-BV510 and -FITC (clone H57-597, Biolegend and BD Biosciences) and SiglecF-PE (clone 1RNM44N, eBioscience).

Techniques: Infection, Flow Cytometry

Journal: PLoS ONE

Article Title: Effects of Exogenous Recombinant APC in Mouse Models of Ischemia Reperfusion Injury and of Atherosclerosis

doi: 10.1371/journal.pone.0101446

Figure Lengend Snippet: Images of plaques stained for collagen ( A ), macrophages ( B ), leukocytes ( C ), and T cells ( D ). E) After sirius red staining, the percentage of collagen was determined by dividing the area of collagen by the total plaque area. F) After Mac-3 staining, the percentage of macrophages as a percentage of the total number of cells per plaque was calculated. G) After CD45 staining, the percentage of leukocytes as a percentage of the total number of cells per plaque was calculated. H) After CD3 staining, the percentage of T cells of the total number of cells per plaque was calculated. All graphs show mean +/− SEM, statistical significance was tested using one-way analysis of variance with Dunnett post hoc test, *P<0.05, **P<0.01.

Article Snippet: Blood and spleen were collected, processed and fluorescently stained with either antibody mix 1 (CD3-FITC, CD4-PerCp, CD8-eFLUO450, CD25-APC, Foxp3-PE and B220-PE-Cy7) or antibody mix 2 (CD11b-PE-Cy7, Ly6C-APC, Ly6G-PE, CD3-FITC, B220-APC-eFLUO780 and NK1.1-PerCp-Cy5.5) or isotype control IgG (all BD Biosciences or eBioscience).

Techniques: Staining