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Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology <t>of</t> <t>isolated</t> exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using <t>ddPCR</t> (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3). For ( E and G ), statistical analysis was performed using Student’s t -test. Significance levels, in comparison to the PBS-exo group, are indicated as follows: * p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
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Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology <t>of</t> <t>isolated</t> exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using <t>ddPCR</t> (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3). For ( E and G ), statistical analysis was performed using Student’s t -test. Significance levels, in comparison to the PBS-exo group, are indicated as follows: * p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
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Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology of isolated exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using ddPCR (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3). For ( E and G ), statistical analysis was performed using Student’s t -test. Significance levels, in comparison to the PBS-exo group, are indicated as follows: * p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Journal: Infection and Drug Resistance

Article Title: Exosomal miR-148a-3p from LPS-Activated Macrophages Promotes M1 Polarization and Ferroptosis-Related Characteristics of Recipient Macrophages by Reducing SLC7A11

doi: 10.2147/IDR.S590998

Figure Lengend Snippet: Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology of isolated exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using ddPCR (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3). For ( E and G ), statistical analysis was performed using Student’s t -test. Significance levels, in comparison to the PBS-exo group, are indicated as follows: * p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: Figure 2 Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology of isolated exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using ddPCR (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3).

Techniques: Isolation, Concentration Assay, Negative Control, Marker, Western Blot, Fluorescence, Imaging, Incubation, Labeling, Staining, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Comparison