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: Glia

Article Title: Activated microglia do not increase 18 kDa translocator protein ( TSPO ) expression in the multiple sclerosis brain

doi: 10.1002/glia.24052

Figure Lengend Snippet: Antibodies for immunohistochemistry

Article Snippet: CD68 , Rabbit , Atlas Antibody , HPA048982 , 1:4000.

Techniques:

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: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: Iba1 + (microglia/monocyte lineage), CD68 + (macrophages) and MPO + (neutrophils) cells presence in the olfactory epithelium before and during SARS-CoV-2 infection. Immunostaining on successive slides of the olfactory epithelium from a non-infected ( A ) or 1 dpi hamster ( B ). Only Iba1 + cells are present in the uninfected olfactory epithelium (OE) and in the lamina propria (LP). In the infected epithelium, Iba1 + cells are massively present in the OE while CD68 + and MPO cells are mostly present in the desquamated cells (red asterisk) in the lumen of the nasal cavity (white asterisk)

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Immunostaining

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: CD68 + macrophage and MPO + neutrophil cells are associated with damage of the olfactory epithelium during SARS-CoV-2 infection. CD68 + ( A 1 ) and MPO + ( B 2 ) signal in the olfactory epithelium (OE, left) and lamina propria (LP, right) in either control animals (CTL) or at 1 or 2 days post-infection (dpi) (Mean normalized to control ± SEM, n = 4, * p < 0.05 (Mann–Whitney test)). Correlation between score damage and percentage of CD68 + ( A 1 ) and MPO + ( B 2 ) signal in the olfactory epithelium (left panel) and the lamina propria (right panel). Spearman test p value

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Control, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: Immunosuppression induced by cyclophosphamide reduces damage of the olfactory epithelium as well as OE infection area. ( A ) Expression of innate immune genes in the nasal turbinates with or without cyclophosphamide treatment at 1 and 2 days post-infection (dpi). Iba1, CD68 and Ncf2 are related to the presence of microglia/macrophages, monocytes/macrophages and neutrophils, respectively; TNFα and IL6 are two cytokines expressed during inflammation; SARS-CoV-2 N expression is related to the SARS-CoV-2 infection. Results represent the Mean ± SEM relative to vehicle-treated hamsters ( n = 4, * p < 0.05; Mann–Whitney test). Representative images of the infected olfactory epithelium immunostained for MPO (neutrophil marker) and SARS-CoV-2 N protein in ( B ) vehicle and ( C ) cyclophosphamide treated animal (olfactory epithelium (OE), lamina propria (LP)). In the vehicle condition, the lumen (white asterisk) is filled with desquamated cells (red asterisk) containing MPO signal. In the cyclophosphamide condition, MPO signal is absent and the lumen is mostly free of cellular debris . Quantification in the OE of ( D 1 ) MPO + neutrophil presence ( D 2 ) damage score ( D 3 ) SARS-CoV-2-infected area and in the lumen of the nasal cavity of ( D 4 ) desquamated cells area and ( D 5 ) percentage of SARS-CoV-2-infected area in the desquamated cells (Mean ± SEM, n = 8 areas of the nasal cavity from 4 different animals, * p < 0.05, ** p < 0.01, *** p < 0.001 (Mann–Whitney))

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Expressing, MANN-WHITNEY, Marker

Journal: Journal of Virology

Article Title: Brain Large Artery Lymphocytic Inflammation and Human Immunodeficiency Virus-Related Brain Arterial Remodeling

doi: 10.1128/JVI.00081-18

Figure Lengend Snippet: Intima CD3 + /CD68 + cell phenotypes and markers of brain arterial remodeling a

Article Snippet: Briefly, anti-CD68 + antibody (primary, 1:100, catalog number M0814, from Dako Corp., Carpinteria, CA; secondary, biotinylated horse anti-mouse IgG antibody, catalog number BA-2000, from Vector Laboratories, Burlingame, CA) binding to arteries was counterstained with hematoxylin and visualized using the diaminobenzidine kit ( 10 ).

Techniques: Isolation