anti-cd14 alexa700  (Bio-Rad)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of Human Afferent Lymph Dendritic Cells from Seroma Fluids

doi: 10.4049/jimmunol.1300760

Figure Lengend Snippet: Identification of DCs in human afferent lymph from seroma fluids. (A and B) CD3+ T cells isolated from seroma, peripheral blood, and the indicated exudates were analyzed for the expression of CCR7, known to be expressed only on naive and central memory T cells. Dot plots shown in (A) are representative of the comparative stainings summarized in (B). (C) MNCs isolated from seroma fluids were stained for CD3, CD19, NKp46, BDCA2 (lineage) and HLA-DR, CD14, CD1a, and CD207. HLA-DRbrightCD1a+, HLA-DRdimCD14+, and CD207+ Langerhans cells were gated as depicted. (D) HLA-DRbright and HLA-DRdim DCs were sorted by flow cytometry and analyzed by optical microscopy. (E) Afferent lymph DCs form strong aggregates with CD3+ T cells. MNCs from seroma fluids were stained for CD3, HLA-DR, and BDCA1. Large and scattered BDCA1+CD3+ events represent DC/T cell aggregates.

Article Snippet: The following mouse anti-human mAbs were used in our study: anti–HLA-DR Pe-Cy7, -CD1b PE, -CD3 FITC, -CD4 PE,-CD4 APC, -CD11b APC, -CD11c PE and APC, -CD16 PE, -CD19 FITC, -CD40 PE, -CD48 PE, -CD62L PE, -CD68 PE, -DEC205, -DC-SIGN, all from BD Biosciences; anti–BDCA-1, –BDCA-2, –BDCA-3, –BDCA-4 PE, anti-CD11c PE and APC, anti-CD14 APC, anti-CD64 PE, -Foxp3 APC, CLEC9A FITC from Miltenyi Biotec; anti-CD4 PeCy7,-CD25 FITC, -CD80 FITC, -CD83 PE, -CD86 PE, -ILT3 FITC, -langerin PE (CD207), from Instrumentation Laboratories; anti-CD1a Pacific Blue, -CD32 PE, -FceRI PE, -CD115 PE, -CD135 PE, -sirp-α PE, -ILT2 PE, -ILT4 PE, -Foxp3 PE from BioLegend; anti-CCR1, -CCR5, -CCR7, -CXCR3 from R&D; anti–PDL-1 PE, –PDL-2 PE, -Tbet Pe-Cy7,-GATA3 FITC, –retinoic acid–related orphan receptor (ROR)gt PE, -RORγt APC from eBiosciences; anti-CD14 Alexa700 (AbD Serotec); and anti-NKp46 (BAB281) and anti-B7H3, kindly provided by Prof. Alessandro Moretta, Genoa, Italy.

Techniques: Isolation, Expressing, Staining, Flow Cytometry, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of Human Afferent Lymph Dendritic Cells from Seroma Fluids

doi: 10.4049/jimmunol.1300760

Figure Lengend Snippet: Phenotype of afferent lymph DC subsets. (A) The indicated leukocyte markers were comparatively analyzed on HLA-DRbright and HLA-DRdim DC subsets. Bars represent mean values and standard deviations of data obtained in 12 seroma samples. (B) DCs contained in seroma fluids are different from both blood circulating DCs and CD14+ cells detectable in exudates. Cell phenotypes comparable to the neoplastic pleural effusions were observed in other inflammatory exudates (not shown). Data shown are representative of analyses performed in pleural effusions from four patients and seroma from eight distinct patients. (C) The HLA-DRbright DC subset contains BDCA3brightCLEC9A+ DCs expressing Sirpα. **p , 0.01. n.s., Not significant; Pleur eff., neoplastic pleural effusion.

Article Snippet: The following mouse anti-human mAbs were used in our study: anti–HLA-DR Pe-Cy7, -CD1b PE, -CD3 FITC, -CD4 PE,-CD4 APC, -CD11b APC, -CD11c PE and APC, -CD16 PE, -CD19 FITC, -CD40 PE, -CD48 PE, -CD62L PE, -CD68 PE, -DEC205, -DC-SIGN, all from BD Biosciences; anti–BDCA-1, –BDCA-2, –BDCA-3, –BDCA-4 PE, anti-CD11c PE and APC, anti-CD14 APC, anti-CD64 PE, -Foxp3 APC, CLEC9A FITC from Miltenyi Biotec; anti-CD4 PeCy7,-CD25 FITC, -CD80 FITC, -CD83 PE, -CD86 PE, -ILT3 FITC, -langerin PE (CD207), from Instrumentation Laboratories; anti-CD1a Pacific Blue, -CD32 PE, -FceRI PE, -CD115 PE, -CD135 PE, -sirp-α PE, -ILT2 PE, -ILT4 PE, -Foxp3 PE from BioLegend; anti-CCR1, -CCR5, -CCR7, -CXCR3 from R&D; anti–PDL-1 PE, –PDL-2 PE, -Tbet Pe-Cy7,-GATA3 FITC, –retinoic acid–related orphan receptor (ROR)gt PE, -RORγt APC from eBiosciences; anti-CD14 Alexa700 (AbD Serotec); and anti-NKp46 (BAB281) and anti-B7H3, kindly provided by Prof. Alessandro Moretta, Genoa, Italy.

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of Human Afferent Lymph Dendritic Cells from Seroma Fluids

doi: 10.4049/jimmunol.1300760

Figure Lengend Snippet: Comparative analysis of the expression of relevant surface molecules in different human myeloid APCs

Article Snippet: The following mouse anti-human mAbs were used in our study: anti–HLA-DR Pe-Cy7, -CD1b PE, -CD3 FITC, -CD4 PE,-CD4 APC, -CD11b APC, -CD11c PE and APC, -CD16 PE, -CD19 FITC, -CD40 PE, -CD48 PE, -CD62L PE, -CD68 PE, -DEC205, -DC-SIGN, all from BD Biosciences; anti–BDCA-1, –BDCA-2, –BDCA-3, –BDCA-4 PE, anti-CD11c PE and APC, anti-CD14 APC, anti-CD64 PE, -Foxp3 APC, CLEC9A FITC from Miltenyi Biotec; anti-CD4 PeCy7,-CD25 FITC, -CD80 FITC, -CD83 PE, -CD86 PE, -ILT3 FITC, -langerin PE (CD207), from Instrumentation Laboratories; anti-CD1a Pacific Blue, -CD32 PE, -FceRI PE, -CD115 PE, -CD135 PE, -sirp-α PE, -ILT2 PE, -ILT4 PE, -Foxp3 PE from BioLegend; anti-CCR1, -CCR5, -CCR7, -CXCR3 from R&D; anti–PDL-1 PE, –PDL-2 PE, -Tbet Pe-Cy7,-GATA3 FITC, –retinoic acid–related orphan receptor (ROR)gt PE, -RORγt APC from eBiosciences; anti-CD14 Alexa700 (AbD Serotec); and anti-NKp46 (BAB281) and anti-B7H3, kindly provided by Prof. Alessandro Moretta, Genoa, Italy.

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of Human Afferent Lymph Dendritic Cells from Seroma Fluids

doi: 10.4049/jimmunol.1300760

Figure Lengend Snippet: Developmental relationship between DC subsets of afferent lymph. (A) HLA-DRbright DCs of afferent lymph resemble CD11clowCD14− CD1a+/bright migratory DCs recently identified in lymph nodes (19, 25). One representative experiment of 25 is shown. (B) CD14+ cells and CD14+CD1a+ cells (both composing the HLA-DRdim DC subset) represent transitional stages of differentiation from CD14+ precursors to CD1a+CD14‒ DCs (HLA-DRbright). CD14+CD1a−, CD14+CD1a+, and CD14−CD1a+ were sorted by flow cytometry and cultured up to 72 h in the presence of autologous T cells and GM-CSF. Although the CD14−CD1a+ DC population (HLA-DRbright) maintained a stable phenotype in vitro, the other two populations progressively lost CD14 and acquired CD1a. Data shown are representative of results from five independent experiments. (C) Seroma fluids were collected once a week until clinically required. In many instances, a single patient required up to seven seroma aspirations. After 7 wk, the surgical wound had completely healed and no sign of inflammation was present. As shown, the composition of DCs changed between early (postsurgical) and late time points of lymph collection, because after 30 d from surgery only HLA-DRbright DCs were almost exclusively detectable in seroma fluids. Dot plots shown are representative of seroma samples collected from 10 patients.

Article Snippet: The following mouse anti-human mAbs were used in our study: anti–HLA-DR Pe-Cy7, -CD1b PE, -CD3 FITC, -CD4 PE,-CD4 APC, -CD11b APC, -CD11c PE and APC, -CD16 PE, -CD19 FITC, -CD40 PE, -CD48 PE, -CD62L PE, -CD68 PE, -DEC205, -DC-SIGN, all from BD Biosciences; anti–BDCA-1, –BDCA-2, –BDCA-3, –BDCA-4 PE, anti-CD11c PE and APC, anti-CD14 APC, anti-CD64 PE, -Foxp3 APC, CLEC9A FITC from Miltenyi Biotec; anti-CD4 PeCy7,-CD25 FITC, -CD80 FITC, -CD83 PE, -CD86 PE, -ILT3 FITC, -langerin PE (CD207), from Instrumentation Laboratories; anti-CD1a Pacific Blue, -CD32 PE, -FceRI PE, -CD115 PE, -CD135 PE, -sirp-α PE, -ILT2 PE, -ILT4 PE, -Foxp3 PE from BioLegend; anti-CCR1, -CCR5, -CCR7, -CXCR3 from R&D; anti–PDL-1 PE, –PDL-2 PE, -Tbet Pe-Cy7,-GATA3 FITC, –retinoic acid–related orphan receptor (ROR)gt PE, -RORγt APC from eBiosciences; anti-CD14 Alexa700 (AbD Serotec); and anti-NKp46 (BAB281) and anti-B7H3, kindly provided by Prof. Alessandro Moretta, Genoa, Italy.

Techniques: Flow Cytometry, Cell Culture, In Vitro