Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome.

doi: 10.7717/peerj.12312

Figure Lengend Snippet: Figure 4 Expression levels of CCR2 and FPR3 in macrophage model of LPS-induced ARDS and pri- mary alveolar macrophages from ARDS patients. (A) Relative mRNA expressions of CCL13, CCR2, CX3CR1, CXCL16, FPR3 and PLAU on THP-1-derived macrophages in response to LPS were assessed us- ing qRT-PCR. (B) The protein levels of CCR2 and FPR3 under a varying time of LPS treatment were eval- uated by Western blot analysis. (C) CCR2 expression on surface of THP-1-derived macrophages after LPS treatment was detected by flow cytometry analysis. (continued on next page...)

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome.

doi: 10.7717/peerj.12312

Figure Lengend Snippet: Figure 5 Assessment of CCR2 and FPR3 in LPS-induced chemotaxis of macrophages in in vitro experiment. (A)Diagrammatic illustration for the chemotaxis assay procedure: Upper:1 ×10 4 of wild-type or siRNA-transfected macrophages were cultured in serum-free medium with or without LPS. Lower: Culture medium with or without CCL2/fMLP. (B) LPS-induced macrophage chemotaxis toward chemokine CCL2 was assessed using a transwell assay as described in the Methods. (C) Likewise, LPS- triggered macrophage migration toward fMLP was evaluated as above. (D) The correlation between CCR2 and FPR3 expression was analyzed using pearson correlation analysis with expression data of GSE89953 and GSE116560. (E) Elevated protein expression of CCR2 induced by LPS stimulation was significantly attenuated by siRNA-FPR3 transfection. Data are presented as mean ± SD and are representative of 3 independent experiments. ∗P < 0.05, by unpaired, two-tailed Student’s t test.

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Chemotaxis Assay, In Vitro, Transfection, Cell Culture, Transwell Assay, Migration, Expressing, Two Tailed Test

Journal: The Journal of allergy and clinical immunology

Article Title: Peptides from allergenic lipocalins bind to formyl peptide receptor 3 in human dendritic cells to mediate T H 2 immunity

doi: 10.1016/j.jaci.2019.07.008

Figure Lengend Snippet: Cytokine production of allergen-treated DCs silenced for FPR3 expression and cocultured CD4 + CD45RA + T cells. A, Supernatants of DCs silenced for FPR3 expression or controls activated with lipocalins, as indicated, were analyzed by means of ELISA for IL-12 p70. B-D, Supernatants of day 5 cocultures of DCs silenced for FPR3 expression and treated with Can f 1 or Fel d 4, as indicated, and naive CD4 + CD45RA + T cells were analyzed for IL-13 (Fig 4, B ), IL-4 (Fig 4, C ), and IFN-γ (Fig 4, D ) content. ns , P >.05, * P <.05, ** P <.01, and *** P <.001.

Article Snippet: Immunostaining was performed with polyclonal rabbit anti-human FPR3 IgG (LSBio, Seattle, Wash), followed by Alexa Fluor 488–conjugated goat antirabbit antibody (Invitrogen Life Technologies, Eugene, Oreg).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of allergy and clinical immunology

Article Title: Peptides from allergenic lipocalins bind to formyl peptide receptor 3 in human dendritic cells to mediate T H 2 immunity

doi: 10.1016/j.jaci.2019.07.008

Figure Lengend Snippet: Effect of WRW4 on FPR3-induced T H 2 bias. A, WRW4 (1 μmol/L) was added before addition of Can f 1 digest and ionomycin to HEK 293–FPR3 cells. B, IL-12 production by DCs treated with allergenic and nonallergenic lipocalins in the presence or absence of 1 μmol/L WRW4, as indicated. C-E, IL-13 (Fig 5, C ), IL-4 (Fig 5, D ), and IFN-γ (Fig 5, E ) content in supernatants of cocultures of CD4 + CD45RA + naive T H cells and DCs treated with allergenic and nonallergenic lipocalins in the presence or absence of WRW4, as indicated. ns, P >.05, ** P <.01, and *** P <.001.

Article Snippet: Immunostaining was performed with polyclonal rabbit anti-human FPR3 IgG (LSBio, Seattle, Wash), followed by Alexa Fluor 488–conjugated goat antirabbit antibody (Invitrogen Life Technologies, Eugene, Oreg).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Strain-specific Loss of Formyl Peptide Receptor 3 in the Murine Vomeronasal and Immune Systems *

doi: 10.1074/jbc.M116.714493

Figure Lengend Snippet: Fpr3 is expressed in vomeronasal sensory neurons and immune cells. A, left, in situ hybridization with Fpr3 sense and antisense probes on coronal slices of the vomeronasal organ. Black triangles mark Fpr3-positive cells. Scale bar, 100 μm. Right, RT-PCR analysis of Fpr3 expression in the vomeronasal organ. A band of the correct size and sequence was observed in VNO cDNA (+RT) but not in the negative control without reverse transcriptase (−RT). Similar results were obtained in five independent experiments. Size marker (L) FastRuler Middle Range DNA Ladder. B, left, representative immunostaining with Fpr3-ECL2 on dissociated vomeronasal cells of C57Bl/6NCrl mice. The white triangle marks a Fpr3-positive cell (red). Right, quantification of Fpr3 immunoreactivity. Average frequency of Fpr3 expression was analyzed in a total of 60,426 cells from six independent experiments. Antibody specificity was demonstrated by blocking the specific binding site through preincubation with 10 μg/ml of the peptide used for antibody generation. C, colocalization of Fpr3 (red) with different cellular markers (green) on dissociated vomeronasal cells from C57Bl/6NCrl mice using phosphodiesterase 4A (PDE4A) as a marker for the apical zone of the VNO, vomeronasal type 2 receptor 2 (V2R2) as a marker for Vmn2r-expressing cells, G protein subunit Gαo as a marker for the basal zone, and OMP as a marker for mature VSNs. D, colocalization of Fpr3 (red) with immune cell markers (green) on dissociated cells. Fpr3 staining colocalized with Ly6G (lymphocyte antigen 6G), a neutrophil marker. No colocalization was seen for CD45R (cluster of differentiation molecule 45R) that is expressed by most immune cells but absent in neutrophils. In all colocalization experiments, the colocalizing cells are marked with a white triangle and non-colocalizing with a white arrow. All cells were counterstained with the nuclear dye Hoechst33342 (blue). Insets show Fpr3-positive cells in a ×3 magnification. Scale bars, 20 μm. The bar chart below each picture denotes the quantification from two to five experiments, analyzing the colocalizations in a total of 7000 to 57,000 cells. Precise numbers are given in the graphs. Fpr3-positive cells that coexpress the marker are labeled by +, and cells not coexpressing the marker are labeled with −. Values in parentheses denote positive versus total cells. Error bars, S.D.

Article Snippet: Fpr3 sense and antisense probes (nucleotide 672–1056) from National Center for Biotechnology Information database, accession number NM_008042.2 plus 153 bp of the 3′-untranslated region were synthesized from a full-length Fpr3 DNA template using a digoxigenin-labeling procedure during in vitro transcription.

Techniques: In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Negative Control, Reverse Transcription, Marker, Immunostaining, Blocking Assay, Binding Assay, Staining, Labeling