Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 and BNP on macrophage polarization in LPS-activated RAW264.7 cells. ( a ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. ( b , c ) Effect of L2 on M1 and M2 macrophage subtype expression. ( d , e ) Effect of BNP on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 or BNP (0, 0.2, 0.4 and 0.8 ng/mL) and isatin (0.1 mM), added 20 min earlier. ( f ) M2/M1 ratio in LPS-activated RAW264.7 cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 ࢤ cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. L2 corresponding group; †† p < 0.01 vs. group unstimulated with LPS.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Flow Cytometry, Expressing, Immunolabeling, Software

Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 on macrophage polarization in LPS-activated RAW264.7 cells after interleukin-10 inhibition. ( a , b ) Effect of interlekin-10 (IL-10) on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS for 24 h followed by 48 h treatment with or without exogenous IL-10 (0, 10 and 20 ng/mL). ( c , d ) Effect of L2 on M1 and M2 macrophage subtype expression after IL-10 inhibition. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 (0, 0.4 or 0.8 ng/mL) and IL-10 inhibitor at 10 µg/mL, added 20 min earlier. ( e ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 − cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$$ p < 0.001 vs. L2 corresponding group.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Inhibition, Expressing, Flow Cytometry, Immunolabeling, Software

Journal: Journal of Clinical Investigation

Article Title: B cells produce pathogenic antibodies and impair recovery after spinal cord injury in mice

doi: 10.1172/jci39780

Figure Lengend Snippet: Figure 4 Unilateral intraspinal microinjection of antibodies purified from SCI mice causes hind limb paralysis and neuropathology. (A and B) A sequence of still video images 1 day after injecting naive (uninjured) mice with control (uninjured) (A) or SCI antibodies (B). One complete step cycle is depicted in both cases. (C) Summary of hind limb function ipsilateral to the site of injection. Scoring is based on the BMS scale (0–5): 0, complete paralysis; 5, plantar stepping during more than 50% of step cycles. Scores above 5 were not considered, as our analyses were restricted to the limb on the injected side only. uninj, uninjured mice. **P < 0.01 versus uninjured; §P < 0.001 versus uninjured, 2-way ANOVA with Bonferroni’s post-hoc test. (D and E) Low- and high-power images from a mouse injected with control (D) or SCI antibodies (E), respectively. Note that intraspinal pathology is only evident in mice receiving SCI antibodies; the asterisk indicates the injection target. (F) Phagocytic microglia/ macrophages (red, anti-CD68) colocalize with axon/neuron pathology (green, anti–200-kDa NFH) at the site of injection in mice receiving SCI antibodies. (G–I) High-power images of boxed region in F. Scale bars: 0.2 mm (D–F); 50 μm (G–I).

Article Snippet: A list of primary antibodies and their final concentrations follows: rat antimouse C1q (0.133 μg/ml, clone 7H8; Abcam), rat anti-mouse CD45R/B220 (0.83 μg/ml, clone RA3-6B2; Serotec), mouse anti-mouse CD68 (1 μg/ml, clone FA-11; Serotec), chicken anti-mouse NFH (1 μg/ml; Aves Labs), mouse anti-MBP (1:40,000, clone SMI 94, mouse ascites; Covance), goat anti-mouse IgG (1 μg/ml, γ-chain specific; Southern Biotech), F(ab′)2 fragments of goat anti-mouse IgG heavy and light chains (1 μg/ml; Jackson ImmunoResearch Laboratories Inc.), and goat anti-mouse IgG F(ab′)2 fragments (1 μg/ml, γ-chain specific; Jackson ImmunoResearch Laboratories Inc.).

Techniques: Microinjection, Purification, Sequencing, Control, Injection

Journal: PLoS ONE

Article Title: Imaging Neuroinflammation In Vivo in a Neuropathic Pain Rat Model with Near-Infrared Fluorescence and 19 F Magnetic Resonance

doi: 10.1371/journal.pone.0090589

Figure Lengend Snippet: A , B show the affected right sciatic nerve from the CCI leg shown in stained with anti-CD68 antibody to reveal the presence of macrophages infiltrating the nerve. C , D CD68 positive cells are not present in the left leg (contralateral to surgery ) of the same CCI animal. E , F show a nerve from a separate CCI animal that also exhibits infiltration of CD68 positive cells. The boxed area is enlarged to reveal the granular cytoplasm (black arrow) of the macrophages, indicative of the presence of the nanoemulsion. Sham surgical sciatic nerve and non-surgical control sciatic nerve do not exhibit any CD68 positive cells ( G , H ) and ( I , J ). The fluorescent images A, C, G, and I were all acquired at the same sitting with the exact same image acquisition parameters. Macrophages grown in cell culture take up the nanoemulsion, exhibited as particles evident by both confocal fluorescence emissions 700–850 nm of NIR label (DiR) ( K , M ) as well as transmitted light ( L , N ).

Article Snippet: In separate immunohistochemical experiments using comparable protocols, the recovered control, sham and CCI sciatic nerves were prepared for examination using mouse anti rat CD68 antibody (MCA341R, AbD Serotech, Raleigh, NC) and Alexa fluor 488 donkey anti mouse secondary antibody (A-21202, Invitrogen, Carlsbad, CA) to assess the presence of macrophages that infiltrate the nerve.

Techniques: Staining, Control, Cell Culture, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Systemic Lipopolysaccharide Challenge Induces Inflammatory Changes in Rat Dorsal Root Ganglia: An Ex Vivo Study

doi: 10.3390/ijms232113124

Figure Lengend Snippet: Nuclear translocation of inflammatory transcription factors nuclear factor interleukin 6 (NF-IL6) and signal transducer and activator of transcription 3 (STAT3) in cultured dorsal root ganglia (DRG) cells. Immunocytochemistry was performed in cultured DRG cells obtained from PBS- and LPS-treated rats to examine activation of inflammatory transcription factors. ( A ) In CD-68-positive macrophages (green) an enhanced nuclear signal of NF-IL6 (red) was detectable in rats treated with LPS ( A.2 ) compared to controls ( A.1 ). Calculating the mean nuclear intensity of the signal in the area of the nucleus (blue), a significant increase is detectable ( A.3 ). ( B ) The STAT3 signal (red) was mainly detectable in MAP-positive neurons (green) of DRG cultures of LPS-treated animals ( B.2 ) and controls ( B.1 ). Calculating the mean nuclear intensity of the signal in the area of neuronal nuclei (blue), a significant difference is detectable ( B.3 ). Bars represent the mean ± SEM. ‘ n ’ represents the number of investigated cells of the respective cell type. Scale bars present 10 µm. **: p < 0.01; ****: p < 0.0001. PBS: phosphate buffered saline, LPS: lipopolysaccharide.

Article Snippet: For immunocytochemical identification of cell types, the following monoclonal antibodies or polyclonal antisera were used: CD-68 for macrophages (mouse anti rat-CD-68; 1:1000; AbD Serotec, Oxford, UK) and microtubule-associated protein 2a+b for neurons (mouse AP-20 anti-MAP2a+b; 1:600; Sigma-Aldrich Chemie GmbH).

Techniques: Translocation Assay, Cell Culture, Immunocytochemistry, Activation Assay, Saline