a549 cell culture conditioned media  (Exosome Diagnostics)

Journal: Nanomaterials

Article Title: Nanomaterial Exposure, Extracellular Vesicle Biogenesis and Adverse Cellular Outcomes: A Scoping Review

doi: 10.3390/nano12071231

Figure Lengend Snippet: Physicochemical characteristics of NMs, exposure conditions, and biological effects observed in the respective experimental models.

Article Snippet: [ ] , Pt NPs 40–50 nm , A549 Cell-culture-conditioned media , Differential centrifugation and ExoQuick; DLS, NTA, TEM, SEM, EXOCET TM , FP, qRT-PCR, ELISA, BCA , Exosomes 90–100 nm , TSG101, CD81, CD63, CD9 , ↑ Exosome biogenesis ↑ Exosome total protein concentration ↑ Concentrations of TSG101, CD9, CD63, and CD81 proteins, Typical morphology and no significant difference in size were observed.

Techniques: In Vitro, In Vivo, Membrane, Expressing, Concentration Assay, Activity Assay, Migration, Clinical Proteomics, Permeability

Journal: Nanomaterials

Article Title: Nanomaterial Exposure, Extracellular Vesicle Biogenesis and Adverse Cellular Outcomes: A Scoping Review

doi: 10.3390/nano12071231

Figure Lengend Snippet: Overview of the effects of NM exposure on EVs secretion and consequent biological outcomes.

Article Snippet: [ ] , Pt NPs 40–50 nm , A549 Cell-culture-conditioned media , Differential centrifugation and ExoQuick; DLS, NTA, TEM, SEM, EXOCET TM , FP, qRT-PCR, ELISA, BCA , Exosomes 90–100 nm , TSG101, CD81, CD63, CD9 , ↑ Exosome biogenesis ↑ Exosome total protein concentration ↑ Concentrations of TSG101, CD9, CD63, and CD81 proteins, Typical morphology and no significant difference in size were observed.

Techniques: Isolation, Centrifugation, In Vitro, In Vivo, Control, Next-Generation Sequencing, Western Blot, Immunohistochemistry, Expressing, Bradford Protein Assay, Refractive Index, Protein Concentration, Migration, Activation Assay, Filtration, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Gene Expression, Activity Assay, Spectroscopy

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: Quantitation and characterization of tumor-cell derived exosomes. NanoSight and ImageStream analyses of A549-derived exosomes determined size, concentration, and characterization. Human lung cancer cells A549, H358, and non-tumor cells HEK293 were cultured in exosome depleted media for 24 h. Exosomes were isolated from cultured supernatant by using differential centrifugation ( A ) Mean size and concentration of A549 cell-derived exosome determined by NanoSight analysis, mean size was recorded as 182.6 nm, 154.22 nm, and 142.7 nm for A549, H358 and HEK293 cells respectively. ( B ) NanoSight data analysis showing the small size of H358 and HEK293 exosomes compared to A549 cell-derived exosomes. ( C ) NanoSight data analysis showing significantly reduced concentration of exosomes produced from 160 mL of culture media collected of p53 null human lung cancer cells H358 compared to A549 and HEK293 cell exosomes. ( D ) Representative Figure of ImageStream analysis to characterize A549 derived exosomes by expression signals of CD63, CD9, TSG-101, CD81, and EpCAM. ( E ) Percentage of total expression of conventional exosomes markers expressed over A549, H358 and HEK293 cell-exosomes respectively * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Quantitation Assay, Derivative Assay, Concentration Assay, Cell Culture, Isolation, Centrifugation, Produced, Expressing

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: ImageStream and flow cytometry analyses quantify time-dependent internalization of tumor cell-derived exosomes by THP-1 cells. THP-1 cells were seeded and differentiated into M0 macrophages upon overnight stimulation with PMA (20–100 ng/mL). M0 macrophages were then co-cultured with PKH-26 stained A549-derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 to 72 h. Image Stream analysis showing Bright field, CD64 + , PKH-26 + , and composite images. ( A ) Time-dependent internalization of exosomes by CD64 + populations assessed by internalization of PKH-26 stained A549-derived exosomes. CD64 + population, without internalization indicates M0 phenotype. ( B ) MATLAB analysis of percentage of PKH-26 + and CD64 + PKH-26 + signals to show time-dependent internalization of exosomes. Fluorescent signals were collected from 300 cells for each time point. ( C ) Representative flow cytometry of exosome internalization analysis at 24 h and 72 h. Group comparisons of 24 h exosome-, 24 h exosome+, and 72h exosome+ were made. After co-culture, cells were prepared for flow cytometry and stained with CD64, CD11b, and PKH-26. The experiment was repeated twice, with three replicates per sample in each experiment. Exosome- sample was used as control ( D ) Percentage of internalization by THP-1 macrophages as CD11b + CD64 + PKH-26 + showing a significant increase of uptake in 72 h compared to 24 h * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Staining, Co-Culture Assay

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: A549 cell-derived exosomes differentiate non-committed M0 macrophages to M2 phenotype. A549 human lung cancer cells were cultured in exosome depleted media for 24 h. Exosomes were isolated from cultured supernatant using differential centrifugation. Exosomes were stained with PKH-26 for overnight. THP-1 cells were seeded and transformed into M0 macrophages upon overnight stimulation with PMA (20–100 ng/mL). M0 macrophages were then co-cultured with PKH-26 stained, A549 derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 to 72 h. ( A ) Representative flow cytometry plot showing in-vitro induction of M2 phenotype (CD11b + CD64 + CD163 + CD206 + ) with A549-derived exosomes. ( B ) Left to right panel-Total percentage of CD163 + macrophages, showing significant increase in CD163 + macrophages that have internalized PKH + exosomes. Total percentage of CD206 + macrophages, showing significant induction in CD206 + macrophages that have internalized PKH + exosomes. Total percentage of M2 macrophages as CD11b + CD64 + CD163 + CD206 + showing significantly induced M2 phenotypes with exosome-positive sample. ( C ) THP-1 cells were seeded and transformed into M0 macrophages upon overnight stimulation with PMA (20 ng/mL). M0 macrophages were then co-cultured with A549 derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 h M2 gene signature, CHI3L1 (Ym1), IL-10, RETNLB (Fizz1), and Arg1 were upregulated on 24 h of A549-derived exosomes treatment, assessed by qRT-PCR in the cells. ( D ) ImageStream analyses showing CD64 + , PKH-26 + , CD206 + CD163 + macrophages before and after internalization of PKH+ exosomes. Left panel shows composite images from several M0 macrophages without exosome internalization. The right panel shows composite images of M0 macrophages with exosome internalization that have polarized to become M2 (CD206 + CD163 + ) ( E ) Time-dependent increase of M2 polarization induced by A549-derived exosomes showing by percentage of CD163 + , CD64 + CD163 + and PKH-26 + CD163 + analyzed by using MATLAB analysis with the signals collected from 300 cells in each time points. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Derivative Assay, Cell Culture, Isolation, Centrifugation, Staining, Transformation Assay, Flow Cytometry, In Vitro, Quantitative RT-PCR

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: Alterations in cellular bioenergetics of macrophages co-cultured with Tumor-derived exosomes by extracellular flux analysis. ( A ) Cellular bioenergetic profiles and ( B ) the cellular bioenergetic parameters (Basal OCR, ATP-linked OCR, Proton Leak, Maximal OCR, Reserve Capacity and Non-Mitochondrial OCR) of M0, M1 and M2 macrophages as determined by sequential injection of oligomycin (Oligo), FCCP, antimycin A (AntiA). Mean values from 6–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M1 macrophages. ( C ) Comparison of the cellular bioenergetic profiles of untreated M0 macrophages and those co-cultured with normal cell (HEK293) or tumor cell (A549)–derived exosomes, and ( D ) quantitation of the bioenergetic parameters of C. Mean values from 6–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M0-co-culture with normal cells-derived exosomes. ( E ) Inhibition of nonmitochondrial OCR in nonpermeabilized macrophages using DPI. Mean values from 4–8 replicates with ± sem. # p < 0.005 compared to M0 macrophages; * p < 0.01 compared to M1 macrophages. ( F ) PMP-sensitive mitochondrial OCR in macrophages. Mean values from 3–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M0-co-culture with normal cells-derived exosomes.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Cell Culture, Derivative Assay, Injection, Comparison, Quantitation Assay, Co-Culture Assay, Inhibition