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: Scientific Reports

Article Title: The isolation and characterization of CTC subsets related to breast cancer dormancy

doi: 10.1038/srep17533

Figure Lengend Snippet: Breast cancer PBMCs were first sorted applying gating parameters to select for DAPI − (4′, 6-diamidino-2-phenylindole)/EpCAM − /CD45 − /CD44 + /CD24 − cells. Cells were then subsequently sorted to obtain uPAR/int β1 subsets containing combinatorial expression of these markers. Antibodies used for flow cytometry and cell sorting were: anti-human CD45-APC-Cy7 (Biolegend, cat # 304015, 1:50 dilution), mouse anti-human EpCAM-PE CD326 (eBiosciences, cat # 12-9326-71, 1:40 dilution), anti-human CD24-PE ML5 (Biolegend, cat # 311106, 1:20 dilution), anti-human CD44-PE-Cy7 IM7 (Biolegend, cat # 103030, 1:20 dilution), mouse anti-human uPAR (CD87)-FITC (AbD Serotec cat # MCA2506FT, 1:10 dilution), anti-human int β1 (CD29)-ApC TS2/16 (Biolegend, cat # 3030008, 1:50 dilution). Cells were confirmed to be CTCs by performing RT-PCR, immunoflurescence and genotyping arrays. Representative images are shown.

Article Snippet: To establish whether subsets of CTCs isolated from the same patient and possessing a combinatorial uPAR/int β1 expression could be expanded in culture, we analyzed blood from patients’ peripheral blood mononuclear cells (PBMCs) employing multi-parametric flow cytometry analysis (FACS, ARIA IID, BD BiosciencesTM) by selecting DAPI − / CD45 − /EpCAM-negative/CD44 + /CD24 − /uPAR/int β1 expression markers to capture four combinatorial subsets (uPAR + /int β1 + , uPAR + /int β1 − , uPAR - /int β1 + , uPAR − /int β1 − ) respectively ( ).

Techniques: Expressing, Flow Cytometry, FACS, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: The isolation and characterization of CTC subsets related to breast cancer dormancy

doi: 10.1038/srep17533

Figure Lengend Snippet: Multiparametric flow cytometry (Six fluorescence channels, ARIA IID system, BD Biosciences ™ ) was applied to select EpCAM-negative/CD45 − /CD44 + /CD24 − CTC followed by DEPArray single-cell isolation to select a combinatorial expression of uPAR (FITC), int β1 (ApC) and human epidermal growth factor receptor-2 (HER-2) (PE). DEPArray ™ (Silicon Biosystems, Inc.) analyses were subsequently performed by Cell Browser TM software. Representative single CTCs captured and isolated by DEPArray ™ are shown. DAPI (ThermoFisher Scientific; cat # D1306) = nuclear staining blue. BF = Brightfield.

Article Snippet: To establish whether subsets of CTCs isolated from the same patient and possessing a combinatorial uPAR/int β1 expression could be expanded in culture, we analyzed blood from patients’ peripheral blood mononuclear cells (PBMCs) employing multi-parametric flow cytometry analysis (FACS, ARIA IID, BD BiosciencesTM) by selecting DAPI − / CD45 − /EpCAM-negative/CD44 + /CD24 − /uPAR/int β1 expression markers to capture four combinatorial subsets (uPAR + /int β1 + , uPAR + /int β1 − , uPAR - /int β1 + , uPAR − /int β1 − ) respectively ( ).

Techniques: Flow Cytometry, Fluorescence, Single-cell Isolation, Expressing, Software, Isolation, Staining