Journal: Movement Disorders

Article Title: Mitochondrial and Clearance Impairment in p. D620N VPS35 Patient‐Derived Neurons

doi: 10.1002/mds.28365

Figure Lengend Snippet: Mitochondrial dysfunction in VPS35 mutant induced pluripotent stem cell (IPSC)‐derived neurons. ( A ) Representative image of mitochondria stained with MitoTracker Green FM and evaluation of mitochondrial size ( B ), average links per mitochondrion ( C ), and average nodes per mitochondrion ( D ) in control (Control 1 and 2) and VPS35 D620N mutant (VPS35 clones 1_1 and 1_2) neurons in culture medium without antioxidants (without B27 and ascorbic acid) for 24 hours (n = 4). ( E ) Mitochondrial membrane potential measured by tetramethylrhodamine ethyl ester (TMRE) mean fluorescence intensity and ( F ) mitochondrial reactive oxygen species measured by MitoSOX mean fluorescence intensity by flow cytometry in control (Control 1 and 2) and VPS35 D620N mutant (VPS35 clones 1_1 and 1_2) neurons in culture medium without antioxidants (without B27 and ascorbic acid) for 4 hours (n = 4). ( G ) Mean average oxygen consumption rate (OCR) of control (Control 1 and 2) and VPS35 D620N mutant (VPS35 clones 1_1 and 1_2) neurons over a time course. Measurement of basal OCR is followed by the addition of oligomycin (oligo) 2 μM final concentration, FCCP 250 nM final concentration, and antimycin A (AA) 5 μM final concentration and rotenone (rot) 5 μM final concentration (n = 4). ( H ) Calculated basal respiration, maximal respiration, spare respiratory capacity, proton leak, ATP production, and non‐mitochondrial oxygen consumption (n = 4). All statistical tests were Mann–Whitney tests to compare groups. Error bars show standard deviation and ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Cells were analyzed with the BD LSRFortessa flow cytometry analyser and the mean fluorescence intensity of each dye was assessed on at least 20,000 single cells by using FlowJo LLC software.

Techniques: Mutagenesis, Derivative Assay, Staining, Clone Assay, Fluorescence, Flow Cytometry, Concentration Assay, MANN-WHITNEY, Standard Deviation

Journal: Movement Disorders

Article Title: Mitochondrial and Clearance Impairment in p. D620N VPS35 Patient‐Derived Neurons

doi: 10.1002/mds.28365

Figure Lengend Snippet: Autophagy enhancement and α‐synuclein knockdown are not sufficient to alleviate mitochondrial dysfunction. ( A,B ) Western blot analysis of Lamp1 ( A ) and α‐synuclein ( B ) of control and VPS35 D620N mutant neurons after 24 hours of treatment with DMSO (50 nM) or rapamycin (25 or 50 nM) (n = 5). Values are normalized to DMSO‐treated line respectively. ( C ) Mitochondrial membrane potential measured by tetramethylrhodamine ethyl ester (TMRE) mean fluorescence intensity, ( D ) mitochondrial reactive oxygen species measured by MitoSOX mean fluorescence intensity, and ( E ) mitochondrial mass measured by MitoTracker Green FM mean fluorescence intensity by flow cytometry in control and VPS35 D620N mutant neurons after 24 hours of treatment with DMSO (50 nM) or rapamycin (25 or 50 nM) (n = 5). Values are normalized to DMSO‐treated Control 1. ( F ) Mitochondrial membrane potential measured by TMRE mean fluorescence intensity and ( G ) mitochondrial reactive oxygen species measured by MitoSOX mean fluorescence intensity by flow cytometry in control and VPS35 D620N mutant neurons transduced with scramble shRNA and shRNA against α‐synuclein (n = 3). Values are normalized to scramble transduced line respectively. All statistical tests were Mann–Whitney tests or one‐way ANOVA followed by Sidak's multiple comparisons tests to compare groups and conditions. Error bars show standard deviation and * P < 0.05; ** P < 0.01. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Cells were analyzed with the BD LSRFortessa flow cytometry analyser and the mean fluorescence intensity of each dye was assessed on at least 20,000 single cells by using FlowJo LLC software.

Techniques: Western Blot, Mutagenesis, Fluorescence, Flow Cytometry, Transduction, shRNA, MANN-WHITNEY, Standard Deviation

Journal: Cell Proliferation

Article Title: FGF ‐2 addition during expansion of human bone marrow‐derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential

doi: 10.1111/cpr.12046

Figure Lengend Snippet: Flow cytometry results after P4 in four different culture conditions: Alpha‐MEM±FGF‐2, DMEM±FGF‐2, n = 6 measurements per group. *P < 0.05. (a) CD13, (b) CD14, (c) CD49 and (d) CD90.

Article Snippet: One‐ and two‐colour cytometry were performed on a FACS Scan ® flow cytometry analyser (BD Biosciences, San Jose, CA, USA) using Cellquest Pro ® Software (BD Biosciences).

Techniques: Flow Cytometry

Journal: Cell Proliferation

Article Title: FGF ‐2 addition during expansion of human bone marrow‐derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential

doi: 10.1111/cpr.12046

Figure Lengend Snippet: Flow cytometry results after P4 in four different culture conditions: Alpha‐MEM±FGF‐2, DMEM±FGF‐2, n = 6 measurements per group. *P < 0.05, **P < 0.01. (a) CD146 (b) STRO‐1 and (c) CD340.

Article Snippet: One‐ and two‐colour cytometry were performed on a FACS Scan ® flow cytometry analyser (BD Biosciences, San Jose, CA, USA) using Cellquest Pro ® Software (BD Biosciences).

Techniques: Flow Cytometry

Journal: Cells

Article Title: Regulatory T Cells Induce Metastasis by Activating Tgf-Β and Enhancing the Epithelial–Mesenchymal Transition

doi: 10.3390/cells8111387

Figure Lengend Snippet: Increased migration of cancer cells and Tregs in both tumors established from Treg-co-cultured B16-BL6 cells or tumors injected with Tregs. To assess the effect of Tregs on the migration of dissociated tumor cells, ( A ) B16-BL6 cells were co-cultured with Tregs for 72 h at co-culture ratios of 1:10 when inadherent Tregs were removed from culture by multiple washing steps. Subsequently, the cells were detached and 5 × 10 5 cells were injected subcutaneously into the right abdomen to establish a tumor or ( B ) subcutaneous tumors in the right abdomen were established using naïve B16-BL6 cells and subsequently injected intratumorally with Tregs (2 × 10 7 cells) three times every other day. The migration profile of cells dissociated from the B16-BL6 tumors was analyzed using a modified transwell migration chamber. The cells that had migrated to the lower filter surface were stained with H & E. The images are representatives of results from 3 independent experiments. Original magnification: ×200. Migrated cells were counted in three randomly selected fields. Data are shown as mean ± SD of results from three independent fields/well. *** p < 0.001 or ** p < 0.01. ( C ) Tumors were collected from mice injected with Tregs at 5 days after the final injection. The populations of Tregs in tumors from mice were analyzed by flow cytometry. Gating was for cluster of differentiation (CD)4 + T cells and analysis for CD25 + and Foxp3 + cells. The data are representative of three independent experiments performed in triplicate. Data points are mean ± SD ( n = 3 per group). *** p < 0.001.

Article Snippet: Samples were analysed using a BD Biosciences BD FACScanto II flow cytometry analyser and FACSDiva software (BD Biosciences).

Techniques: Migration, Cell Culture, Injection, Co-Culture Assay, Modification, Staining, Flow Cytometry