Journal: Stem Cells Translational Medicine

Article Title: Laminin-111 Improves Skeletal Muscle Stem Cell Quantity and Function Following Eccentric Exercise

doi: 10.5966/sctm.2014-0044

Figure Lengend Snippet: LM-111 does not enhance muscle-resident mesenchymal stem cell (mMSC) content following eccentric exercise. (A): Quantitation of total number of Sca-1+CD45− mMSCs. (B): Quantitation of the percentage of Sca-1+CD45− mMSCs. (C): Gastrocnemius-soleus muscle weight. (D): Quantification of mMSC percentage normalized to muscle weight (n = 6 per group). The values are presented as means ± SEM. Abbreviations: EX, exercise group; LM-111, laminin-111; SA, saline; SED, sedentary group.

Article Snippet: Cells were then stained in a cocktail of monoclonal anti-mouse antibodies: Sca-1-PE (600 ng per 10 6 cells) and CD45-APC (300 ng per 10 6 cells) (eBioscience) diluted in 2% FBS in PBS.

Techniques: Quantitation Assay

Journal: Stem Cells Translational Medicine

Article Title: Laminin-111 Improves Skeletal Muscle Stem Cell Quantity and Function Following Eccentric Exercise

doi: 10.5966/sctm.2014-0044

Figure Lengend Snippet: LM-111 improves muscle-resident mesenchymal stem cell (mMSC) gene expression. Quantitative polymerase chain reaction analysis of Sca-1+CD45− mMSC gene expression collected immediately postsorting. The graphs represent ΔΔCT values expressed relative to SA/SED. (A–D): Relative cytokine expression: IL-6, TNFα, IL-1rn, and IL-10. (E): Relative HGF expression. (F): Relative IGF-1 expression. #, p < .05 compared with SA/EX; §, Main effect of LM-111 (n = 5–6 per group). The values are presented as means ± SEM. Abbreviations: EX, exercise group; HGF, hepatocyte growth factor; IGF, insulin growth factor; IL, interleukin; LM-111, laminin-111; SA, saline; SED, sedentary group; TNFα, tumor necrosis factor α.

Article Snippet: Cells were then stained in a cocktail of monoclonal anti-mouse antibodies: Sca-1-PE (600 ng per 10 6 cells) and CD45-APC (300 ng per 10 6 cells) (eBioscience) diluted in 2% FBS in PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Sprint Interval and Sprint Continuous Training Increases Circulating CD34 + Cells and Cardio-Respiratory Fitness in Young Healthy Women

doi: 10.1371/journal.pone.0108720

Figure Lengend Snippet: ISHAGE gating strategy for CAC enumeration. Cells positive for CD45 and therefore leukocytes were gated in R1. Cells in R1 that were positive for CD34 and had low side scatter characteristics were plotted in R2 and defined as CD34 + cells. Cells from R2 were gated on a further plot to determine the CD45 dim population. Subsequently cells from R3 were back-gated from R4 and gated on a further plot (R5) to define CD34 + CD45 dim cells that are located within the lymphocyte population.

Article Snippet: The sample was incubated with 20 μl of Fc-receptor blocker (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min, before incubation with fluorochrome-conjugated antibodies, CD34-PE (10 μl) and CD45-FITC (10 μl; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: PLoS ONE

Article Title: Sprint Interval and Sprint Continuous Training Increases Circulating CD34 + Cells and Cardio-Respiratory Fitness in Young Healthy Women

doi: 10.1371/journal.pone.0108720

Figure Lengend Snippet: * indicates a significant pre to post-training difference in both groups (time effect p <0.05). a) CD34 + cells increased following both SIT (n = 6) and SCT (n = 5; main time effect p = 0.02) with no group x time interaction ( p = 0.83). However, b) CD34 + /CD45 dim cells did not change following either type of training (main time effect p = 0.21; group x time interaction p = 0.67).

Article Snippet: The sample was incubated with 20 μl of Fc-receptor blocker (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min, before incubation with fluorochrome-conjugated antibodies, CD34-PE (10 μl) and CD45-FITC (10 μl; Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: Cells

Article Title: Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

doi: 10.3390/cells8090998

Figure Lengend Snippet: rVAR2 specifically binds to glioma cells and enables their retrieval from blood. ( A ) rVAR2 stains glioma cell lines in a background of white blood cells (WBCs). Res529 (left), KNS-42 (middle) and U87mg (right) cells were mixed with WBCs and stained with V5-tagged rVAR2 in combination with a CF488-conjugated anti-V5 antibody (green), PE-conjugated anti-CD45 and anti-CD66b antibodies (red), and DAPI (blue). Scale bar, 50 µm. ( B ) Flow cytometry analysis showing WBCs (100 µL RBC lysed blood) mixed with U87mg (50,000) cells and detected with either 200 nM rVAR2 and a FITC-conjugated anti-V5 antibody or with a FITC-conjugated anti-V5 antibody alone (control). ( C ) Same as in ( B ) but with no U87mg cells added. ( D ) Recovery of CellTracker Green-stained glioma cells from blood. 100 cells were spiked into 3 mL blood and recovered using rVAR2-coupled beads. Cells were stained with DAPI and scanned on the Cytation 3 Imager. Each dot represents the percentage of recovered cells from one sample. Bars represent mean recoveries and error bars show +/− standard deviation (n ≥ 2) (one-way ANOVA with Bonferroni correction). ( E ) Recovery of CellTracker Green-stained U87mg cells from blood. The indicated number of U87mg cells was spiked into 3 mL blood and captured using rVAR2-coupled beads. Enumeration of cells and data presentation were done as in ( D ).

Article Snippet: The cells were then incubated with a mixture of 200 nM fluorophore-conjugated (Oregon Green ® 488) rVAR2 and PE-labelled anti-CD45 [5B1] (Cat. no. 170-078-081, MACS Miltenyi Biotec, 1:10) and PE-labelled anti-CD66b [REA306] (Cat. no. 130-104-396, MACS Miltenyi BioTec, 1:20) antibodies for 30 min. at room temperature and then washed once in PBS with 2% FBS to remove excess staining reagents.

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: Cells

Article Title: Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

doi: 10.3390/cells8090998

Figure Lengend Snippet: Evaluation of protein pull-down hits from mass spectrometry by proximity ligation assay (PLA). ( A ) Representative images of PLA assays on U87mg, KNS-42, and U118mg cells showing co-localization between rVAR2 and a panel of CSPGs as red dots. Cells were counterstained with DAPI (blue) and analyzed by confocal microscopy. All of the images are shown in same magnification using a 60× objective. ( B ) Quantification of the PLA co-localization signals between rVAR2 and each of the CSPGs analyzed. Data is shown as the number of signals per cell. Red bars represent the mean number of signals per cell. ( C ) Representative image showing specific CSPG4 staining (green) of an rVAR2-captured U87mg cell in a background of WBCs stained for CD45 and CD66b (both red). Cells were stained with DAPI (blue) and visualized on the Cytation 3 Imager with a 20× objective. Scale bar, 50 µm.

Article Snippet: The cells were then incubated with a mixture of 200 nM fluorophore-conjugated (Oregon Green ® 488) rVAR2 and PE-labelled anti-CD45 [5B1] (Cat. no. 170-078-081, MACS Miltenyi Biotec, 1:10) and PE-labelled anti-CD66b [REA306] (Cat. no. 130-104-396, MACS Miltenyi BioTec, 1:20) antibodies for 30 min. at room temperature and then washed once in PBS with 2% FBS to remove excess staining reagents.

Techniques: Mass Spectrometry, Proximity Ligation Assay, Confocal Microscopy, Staining

Journal: Cells

Article Title: Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

doi: 10.3390/cells8090998

Figure Lengend Snippet: Using rVAR2 to stain glioma cells after capture with rVAR2-coupled beads. ( A ) Glioma cell lines (U87mg, Res259, and KNS-42) were incubated with rVAR2-coupled beads and stained with a fluorophore-conjugated rVAR2 (green) and DAPI (blue). Representative images were obtained using the Cytation 3 Imager with a 10× objective. Scale bars, 20 µm. ( B ) U87mg cells and GBM02 cells were spiked into 3 mL blood, retrieved using rVAR2-coupled beads, and stained using fluorescent rVAR2 (green), anti-CD45/CD66b antibodies (red) and DAPI (blue). Scale bars, 20 µm.

Article Snippet: The cells were then incubated with a mixture of 200 nM fluorophore-conjugated (Oregon Green ® 488) rVAR2 and PE-labelled anti-CD45 [5B1] (Cat. no. 170-078-081, MACS Miltenyi Biotec, 1:10) and PE-labelled anti-CD66b [REA306] (Cat. no. 130-104-396, MACS Miltenyi BioTec, 1:20) antibodies for 30 min. at room temperature and then washed once in PBS with 2% FBS to remove excess staining reagents.

Techniques: Staining, Incubation

Journal: Cells

Article Title: Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

doi: 10.3390/cells8090998

Figure Lengend Snippet: rVAR2 enables capture and detection of glioma circulating tumor cells (CTCs) from patient blood samples. ( A ) Average CTC count in blood samples from ten glioma patients. 3 mL blood samples were processed by using rVAR2-coupled beads followed by staining using a mixture of fluorophore-conjugated rVAR2 (green), anti-CD45/CD66b antibodies (red) and DAPI (blue). CTCs were defined as rVAR2+, CD45/CD66b−, DAPI+ cells. Each dot represents the average number of detected CTCs per 3 mL patient blood sample. The x-axis shows whether the patient was diagnosed with GBM, Anaplastic Oligodendroglioma (ODG), or Oligodendroglioma (ODG). ( B ) Representative images of identified CTCs from a patient diagnosed with GBM. The sample was stained with flurophore-conjugated rVAR2 (green), anti-CD45 antibody (magenta), and DAPI (blue). Images were obtained using the CellCelector™ (ALS) with a 40× objective. Scale bars, 20 µm.

Article Snippet: The cells were then incubated with a mixture of 200 nM fluorophore-conjugated (Oregon Green ® 488) rVAR2 and PE-labelled anti-CD45 [5B1] (Cat. no. 170-078-081, MACS Miltenyi Biotec, 1:10) and PE-labelled anti-CD66b [REA306] (Cat. no. 130-104-396, MACS Miltenyi BioTec, 1:20) antibodies for 30 min. at room temperature and then washed once in PBS with 2% FBS to remove excess staining reagents.

Techniques: Staining

Journal: Cells

Article Title: Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

doi: 10.3390/cells8090998

Figure Lengend Snippet: Identification of CTCs for whole exome sequencing (WES). Representative images of identified CTCs from three patients. Cells were stained with fluorophore-conjugated rVAR2 (green), anti-CD45 antibody (magenta) and DAPI (blue) and classified as CTCs if rVAR2+, CD45−, and DAPI+. Images were obtained using the CellCelector™ (ALS) with a 10× objective. Scale bars, 50 µm.

Article Snippet: The cells were then incubated with a mixture of 200 nM fluorophore-conjugated (Oregon Green ® 488) rVAR2 and PE-labelled anti-CD45 [5B1] (Cat. no. 170-078-081, MACS Miltenyi Biotec, 1:10) and PE-labelled anti-CD66b [REA306] (Cat. no. 130-104-396, MACS Miltenyi BioTec, 1:20) antibodies for 30 min. at room temperature and then washed once in PBS with 2% FBS to remove excess staining reagents.

Techniques: Sequencing, Staining