EV samples were adsorbed onto a formvar/carbon-coated nickel grid for 1 h. EVs were fixed with 2% paraformaldehyde and then incubated with the following primary antibodies: anti-CD9 (MM2/57; Thermo Fisher Scientific), anti-CD14 [EPR3653] (ab133335; Abcam, Cambridge, UK) and anti-lipopolysaccharide-binding protein (LBP) polyclonal (PA5-21642; Invitrogen, USA). Immunoreactive EVs were visualized using anti-mouse IgG(H+L) (EMGMHL10) and anti-rabbit IgG(H+L) (EMGFAR10; BBI Solutions, UK) antibodies preabsorbed with 10-nm gold particles.
Emgfar10
Manufactured by BBI Solutions
Sourced in United Kingdom
The EMGFAR10 is a laboratory equipment product from BBI Solutions. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach cannot be provided. Further details on the intended use of this product are not available.
Automatically generated - may contain errors
2 protocols using emgfar10
1
Characterization of Extracellular Vesicles
2
Characterization of Extracellular Vesicles from LPS-Stimulated Macrophages
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Samples of EVs (10 μg) were adsorbed onto a formvar/carbon-coated nickel grid for 1 h. EVs were xed with 2% paraformaldehyde and then incubated with the following primary antibodies: anti-CD9 (MM2/57; Thermo Fisher Scienti c), anti-CD14 [EPR3653] (ab133335; Abcam, Cambridge, UK), and antilipopolysaccharide-binding protein (LBP) polyclonal (PA5-21642; Invitrogen, USA). Immunoreactive EVs were visualized using anti-mouse IgG(H+L) (EMGMHL10) and anti-rabbit IgG(H+L) (EMGFAR10; BBI Solutions, UK) antibodies preabsorbed with 10-nm gold particles.
Cell Culture and Lipopolysaccharide Stimulation. RAW 264.7 , a murine macrophage/monocyte lineage cell line, obtained from ATCC (ATCC no.: TIB-71), was cultured in DMEM containing 0.5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. For stimulation with lipopolysaccharide (LPS), RAW 264.7 cells (1×10 6 cells/ml) were seeded into 6-well plates in DMEM with exosome-free FBS and stimulated with 10 ng/ml LPS for 6 h. EVs in the supernatant (650 μl/well) were collected using size exclusion chromatography column. EVs were then concentrated by ultracentrifugation (100,000×g, 4°C, 70 min), dissolved in RIPA buffer, and evaluated by blotting. RAW 264.7 cells were xed and stained with Diff-Quick stain (Sysmex, Kobe, Japan).
Cell Culture and Lipopolysaccharide Stimulation. RAW 264.7 , a murine macrophage/monocyte lineage cell line, obtained from ATCC (ATCC no.: TIB-71), was cultured in DMEM containing 0.5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. For stimulation with lipopolysaccharide (LPS), RAW 264.7 cells (1×10 6 cells/ml) were seeded into 6-well plates in DMEM with exosome-free FBS and stimulated with 10 ng/ml LPS for 6 h. EVs in the supernatant (650 μl/well) were collected using size exclusion chromatography column. EVs were then concentrated by ultracentrifugation (100,000×g, 4°C, 70 min), dissolved in RIPA buffer, and evaluated by blotting. RAW 264.7 cells were xed and stained with Diff-Quick stain (Sysmex, Kobe, Japan).
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