culture media solutions  (PromoCell)

Journal: Frontiers in Immunology

Article Title: Activated hepatic stellate cell-derived small extracellular vesicles facilitate M2 macrophage polarization and hepatoma progression via miR-27a-3p

doi: 10.3389/fimmu.2024.1489679

Figure Lengend Snippet: miR-27a-3p expression was increased in cells and sEVs of activated HSCs and aLX2 cells vs. their quiescent counterparts. (A–C) NTA, TEM, and western blotting analysis of sEVs isolated from 3 day quiescent HSCs and 14 day activated HSCs. NTA instrument (NanoSight NS300) was calibrated with 100 nm standard. The expression of the characteristic sEVs molecules CD63 and CD81 was detected by western blotting. (D) qRT-PCR was used to detect the expression of miR-27a-3p in cells and sEVs from quiescent (day 3) activated primary rats HSCs (day 11 and day 14). U6 snRNA served as internal reference for cell samples and cel-miR-39 served as external reference for sEVs samples (mean ± SEM). ***P < 0.001, ****P < 0.0001 vs. Control; (E, F) The expression of α-SMA in LX2 cells treated with 10 ng/ml TGFβ1 for 24 h for mRNA as detected by qRT-PCR normalized to GAPDH; or 48 h for protein as detected by western blotting normalized to β-actin (mean ± SEM). ****P < 0.0001 vs. Control; (G–I) NTA, TEM, and western blotting analysis of sEVs isolated from activated LX2 cells. NTA instrument was calibrated with 100 nm standard. The expression of the characteristic sEVs molecules CD63 and CD81 was detected by western blotting; (J) qRT-PCR was used to detect the expression of miR-27a-3p in qLX2 and aLX2 cells and supernatant sEVs in LX2-activation models. U6 snRNA served as internal reference for cell samples and cel-miR-39 served as external reference for sEVs samples (mean ± SEM). ****P < 0.0001 vs. control. 3d, 3 days; 11d, 11 days; 14d, 14 days; qLX2, quiescent LX2; aLX2, activated LX2. Data were from three independent tests.

Article Snippet: To increase the recovery rate, the concentrated mediums were mixed well with Tissue Culture Media SEVs Precipitation Solution (System Biosciences, Inc., USA) and incubated overnight in the dark at 4°C ( ).

Techniques: Expressing, Western Blot, Isolation, Quantitative RT-PCR, Control, Activation Assay

Journal: Frontiers in Immunology

Article Title: Activated hepatic stellate cell-derived small extracellular vesicles facilitate M2 macrophage polarization and hepatoma progression via miR-27a-3p

doi: 10.3389/fimmu.2024.1489679

Figure Lengend Snippet: The miR-27a-3p over-expressing HSC-sEVs promote HepG2 cell migration directly or through educating macrophages. (A) Schematic of the conditioned culture experiments. (B) Representative images for the size distribution and concentration of sEV particles in the supernatant of transfected LX2 cells as detected by NTA. (C) The expression of miR-27a-3p in mimic-miR-27a-3p or an-ti-miR-27a-3p transfected LX2 cells and sEVs was detected by qRT-PCR, standardized to U6 snRNA for cell samples or cel-miR-39 for sEVs samples, respectively. *P < 0.05, ****P < 0.0001 vs. control. ####P < 0.0001 vs. NC. (D, E) Transwell migration assays for conditioned cultured. Representative images for the crystal violet staining of migrated cells, bar = 200 μm; and the number of migrated cells per 100 × field for each group (mean ± SEM). ****P < 0.0001 vs. control. ###P < 0.001, ####P < 0.0001 vs. NC. (F) The expression levels of surface markers in THP1 macrophages was detected using qRT-PCR. *P < 0.05, **P < 0.01 vs. control. #P < 0.05 vs. NC. H, HepG2 cells; qLX2, quiescent LX2 cells; aLX2, active LX2 cells; CM, culture medium. The data were from three independent experiments.

Article Snippet: To increase the recovery rate, the concentrated mediums were mixed well with Tissue Culture Media SEVs Precipitation Solution (System Biosciences, Inc., USA) and incubated overnight in the dark at 4°C ( ).

Techniques: Expressing, Migration, Concentration Assay, Transfection, Quantitative RT-PCR, Control, Cell Culture, Staining

Journal: Frontiers in Immunology

Article Title: Activated hepatic stellate cell-derived small extracellular vesicles facilitate M2 macrophage polarization and hepatoma progression via miR-27a-3p

doi: 10.3389/fimmu.2024.1489679

Figure Lengend Snippet: The interaction between miR-27a-3p/SPRY2 and hepatoma progression. miR-27a-3p was significantly upregulated in HSC-sEVs during the activation of HSCs. HSC-sEVs containing miR-27a-3p could promote M2 polarization of macrophage and proliferation of hepatoma cells by targeting SPRY2, leading to the progression of hepatocellular carcinoma.

Article Snippet: To increase the recovery rate, the concentrated mediums were mixed well with Tissue Culture Media SEVs Precipitation Solution (System Biosciences, Inc., USA) and incubated overnight in the dark at 4°C ( ).

Techniques: Activation Assay