Journal: Journal of Tissue Engineering

Article Title: Continuous nutrient supply culture strategy controls multivesicular endosomes pathway and anti-photo-aging miRNA cargo loading of extracellular vesicles

doi: 10.1177/20417314231197604

Figure Lengend Snippet: Characterization and production: comparison of EVs: (a) isolation method of EVs, (b) TEM images of EVs, (c) particles size distribution, (d) western blot analysis of the particles to detect the expression of EV protein markers (CD81, CD63, CD9 and TSG101), (e) protein concentration of EV suspensions, (f) the number of EV pellets derived from each cell, (g) EV particle concentration, and (h) the size distribution of CCEVs and SC-EVs. Data are expressed as mean plus and minus standard deviations ( n = 3), *** p < 0.001.

Article Snippet: Finally, the total protein extract (40 μg) was separated and the protein expression of Protein 1A/1B-light chain I/II (LC3 I/II), PARP (ZenBioScience, China), CD9, CD63 and CD81 (Abcam, UK) were examined according to the previous method.

Techniques: Comparison, Isolation, Western Blot, Expressing, Protein Concentration, Derivative Assay, Concentration Assay

Journal: Journal of Tissue Engineering

Article Title: Continuous nutrient supply culture strategy controls multivesicular endosomes pathway and anti-photo-aging miRNA cargo loading of extracellular vesicles

doi: 10.1177/20417314231197604

Figure Lengend Snippet: Differential transport of tetraspanins in PM affects the formation of precursors of EVs (Intracellular vesicles content is equal to the whole membrane content minus cells surface membrane content): (a) immunofluorescence flow cytometry of tetraspanins (CD9, CD63, CD81) in PM of SC-MSCs and (b) CCMSCs, (c) expression of tetraspanins in whole cell lysates, (d) quantitative analysis of gray values, (e) images of cell sections captured in different culture methods by TEM, the upper and lower scale bar separately represents 2 μm and 500 nm, and (f) the number of MVEs of MSCs cultured in different ways, the budding ILVs within each MVE, and the membrane surface area of each MVE. Data are expressed as mean plus and minus standard deviations, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Finally, the total protein extract (40 μg) was separated and the protein expression of Protein 1A/1B-light chain I/II (LC3 I/II), PARP (ZenBioScience, China), CD9, CD63 and CD81 (Abcam, UK) were examined according to the previous method.

Techniques: Membrane, Immunofluorescence, Flow Cytometry, Expressing, Cell Culture

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: ( A ) HepG2 cells were incubated with 30 μM berberine at 37°C for 0.5, 1, 2, 4, or 6 h, respectively. ( B ) HepG2 cells were incubated with 0.9, 3, 9, 30, 90, or 300 μM berberine at 37°C for 0.5 h, respectively. ( C ) HepG2 cells were incubated with 0.9, 3, 9, 30, 90, or 300 μM berberine at 4 or 37°C for 0.5 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the accumulated berberine in the cells was semi-quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Flow Cytometry

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: ( A ) HepG2 cells were incubated with HBSS or HBSS-K containing 10 μM berberine at 37°C for 0.5 h, respectively. ( B ) HepG2 cells were incubated with 10 μM berberine or in the presence of cimetidine (0.3, 1 mM, Cim) or rifampicin (10 μM, Rif) at 37°C for 0.5 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the accumulated berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. ** indicates P <0.01 versus control or between 0.3 or 1 mM cimetidine-treated groups.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Flow Cytometry, Control

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: HepG2 cells were pre-incubated with drug-free HBSS or HBSS containing 0.125, 0.25, 0.5, 1, or 2 μM CCCP at 37°C for 20 min, then equal volume of HBSS containing 20 μM berberine or 6 μg/ml Rho123 was added and the cells were further incubated at 37°C for 0.5 h. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in chilled PBS and the concentration of berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. * indicates P <0 . 05 while ** indicates P <0.01 versus groups that treated without CCCP.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Concentration Assay, Flow Cytometry

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: HepG2 cells were incubated with 10 μM berberine and with or without 0.125 μM CCCP, 0.3 or 1 mM cimetidine (Cim), or the HBSS-K solution 37°C for 0.5 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the concentration of berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. ( A ) Synergistic inhibition of CCCP and HBSS-K on berberine uptake in the HepG2 cells; ( B ) synergistic inhibition of HBSS-K and cimetidine on berberine uptake in the HepG2 cells; ( C ) influence of CCCP on the inhibition of HBSS-K in terms of berberine uptake in the HepG2 cells; ( D ) inhibition of the combination of CCCP, HBSS-K, and cimetidine on berberine uptake in the HepG2 cells.* indicates P <0 . 05 while ** indicates P <0.01 versus control or between groups indicated by the lines.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Concentration Assay, Flow Cytometry, Inhibition, Control

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: HepG2 cells were pre-incubated with 10 μM berberine at 37°C for 2 h, then the cells were incubated with drug-free HBSS or HBSS containing CCCP (0.25 μM), or verapamil (100 μM), or CCCP (0.25 μM) plus verapamil (100 μM) at 37°C for 0.5, 1, 2, 4 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the accumulated berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. The amount of berberine was normalised to that at zero time point. * indicates P <0.05 while ** indicates P <0.01 versus verapamil treated groups.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Flow Cytometry