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: American Journal of Respiratory Cell and Molecular Biology

Article Title: Mesenchymal Stem Cell Extracellular Vesicles Reverse Sugen/Hypoxia Pulmonary Hypertension in Rats

doi: 10.1165/rcmb.2019-0154OC

Figure Lengend Snippet: (A) Immunohistochemical staining of Cd206+ macrophages in lungs of rats treated with normoxia, (B and C) SuHx plus vehicle (B), and SuHx plus MSC-EV (C). Scale bars: 50 μm. (D–F) Average number of Cd206+ per 10× field (D), Cd68+ total rat macrophages per 10× field (E), and ratio of Cd206+/Cd68+ macrophages per 10× lens objective field of view (F) in lung sections from rats treated with Nx, SuHx plus vehicle, or SuHxEV. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: Lungs were kept in 4% paraformaldehyde until embedded in paraffin; sectioned in 5-μm slices; and stained with antibody against rat ACTA2 (ab5694; Abcam) to assess pulmonary vascular remodeling, antibody against rat von Willebrand factor (vWF) (ab6994; Abcam) to assess distal vessel count, and antibodies against rat Cd206 (ab64693; Abcam) or Cd68 (ED1; Bio-Rad Laboratories) for assessment of macrophage recruitment.

Techniques: Immunohistochemical staining, Staining

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