Journal: Cancer Science

Article Title: Synthetic disulfide-bridged cyclic peptides mimic the anti-angiogenic actions of chondromodulin-I

doi: 10.1111/j.1349-7006.2012.02276.x

Figure Lengend Snippet: Effects of the synthetic and tailed chondromodulin-I (ChM-I) cyclic peptide on tumor angiogenesis and growth in an animal model in which OUMS-27 cells (5 × 10 6 cells) were subcutaneously inoculated in the backs of 4 week-old nude mice. (A) Time-course of tumor volume changes. When the tumor volume reached about 45 mm 3 , each mouse was injected around the tumor mass each day for the initial 5 days with PBS (50 μL) alone (♢), PBS containing 4.3 nmol (20 μg) ChM-I cyclic peptide with a tail (○), or PBS containing 0.2 nmol (5 μg) recombinant human ChM-I (rhChM-I) (●) (arrows). The tumor volumes were determined by the width 2 × length × 0.52. Values are the means ± SD from at least six animals per group. (B) Gross appearance of tumors excised on day 21. Bar, 10 mm. (C) Immunohistochemical staining of the CD31-positive vasculature. On day 21, tumor tissues were excised, fixed, and cross-sectioned. The sections were then stained with toluidine blue (left panels), and semi-serial sections were stained with an anti-type II collagen antibody ( green signal in the right panels) and an anti-CD31 antibody ( red signal in the right panels), respectively. Bars, 100 μm. (D) The CD31-positive area was measured as described in the methods section. Values are the means ± SD of five tumors per group. * P < 0.05, ** P < 0.01.

Article Snippet: Anti-CD31 antibody and anti-collagen type II antibody were obtained from BD PharMingen (San Diego, CA, USA) and Rockland (Gilvertsville, PA, USA), respectively.

Techniques: Animal Model, Injection, Recombinant, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia

doi: 10.1038/s41598-018-28211-5

Figure Lengend Snippet: MC enables an order of magnitude more parameters than FFC to be analyzed simultaneously during platelet activation. Citrate-anticoagulated blood from the same 3 separate healthy donors in Fig. was simultaneously treated with vehicle or the indicated concentrations of TRAP ( A ) or ADP ( B ) for 30 minutes in the presence of a custom platelet-specific, metal-tagged antibody panel. This panel contained antibodies directed against CD41, CD61, CD63, CD9, CD107a, CD154, CD42a, CD42b, CD31, CD36, CD29 and GPVI. Samples were fixed in 1% formaldehyde and analyzed by MC. Data were analyzed using a non-linear fit of log agonist vs . response; variable slope or linear regression in GraphPad Prism 5. Results are expressed as a percentage of the mean metal intensity (MMI) achieved with 200 µM ADP or TRAP (means ± SEM; n = 2/3, with n = 2 accounting for the linear region of the TRAP dose-response [concentrations 1.5–3.5 µM)] and n = 3 accounting for all other concentrations [0–1 µM and 5–200 µM]). Statistical analysis: 1-way ANOVA was used in conjunction with a Dunnett multiple comparison test (all results compared to vehicle control) to indicate statistical significance; *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: ADP, adenosine diphosphate; FFC, fluorescence flow cytometry; MC, mass cytometry; TRAP, thrombin receptor activating peptide.

Article Snippet: Metal-conjugated monoclonal antibodies were from Fluidigm Corporation (San Francisco, CA): anti-CD9-171Yb (clone SN4 C33A2), anti-CD31-145Nd (clone WM59), anti-CD61-165Ho (clone VI-PL2); from Longwood Medical Area Antibody Core (Boston, MA): anti-CD36-150Nd (clone 5-271), anti-CD42b-163Dy (clone HIP1), anti-CD41-149Sm (clone HIP8), anti-CD62P-172Yb (clone AK4), anti-CD63-161Dy (clone H5C6), anti-CD107a-166Er (clone H4A3), anti-CD154-154Sm (clone 24-31); or labeled in-house (described below): anti-CD29-176Yb (Biolegend, San Diego, CA, clone TS2/16), anti-CD42a-155Gd (BD Biosciences, San Jose, CA, clone ALMA.16), anti-GPVI-152Sm (EMD Millipore, Billerica, MA, polyclonal IgG), anti-activated αIIbβ3 (PAC1)-159Tb (BD Biosciences, San Jose, CA, clone PAC1).

Techniques: Activation Assay, Fluorescence, Flow Cytometry, Mass Cytometry

Journal: Scientific Reports

Article Title: Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia

doi: 10.1038/s41598-018-28211-5

Figure Lengend Snippet: Multidimensional analysis of platelet subpopulations by MC reveals heterogeneity in healthy donor samples. Visual stochastic neighbor embedding (viSNE) plots of whole blood samples drawn on 3 separate days from the same healthy subject (a different healthy subject from the healthy subjects analyzed in Figs and ). Samples were stained with a metal-tagged antibody cocktail containing 12 markers (directed against: CD9, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154 and activated integrin αIIbβ3), treated with vehicle or 20 µM TRAP, and analyzed using MC. Color intensity relates to antigen expression (low [blue] or high [red]) and each dot represents an individual platelet. The distance between dots/platelets and populations of dots/platelets is inversely proportional to how closely related those dots/platelets are in terms of antigen expression and characteristics. Abbreviations: TRAP, thrombin receptor activating peptide; tSNE, t-distributed stochastic neighbor embedding.

Article Snippet: Metal-conjugated monoclonal antibodies were from Fluidigm Corporation (San Francisco, CA): anti-CD9-171Yb (clone SN4 C33A2), anti-CD31-145Nd (clone WM59), anti-CD61-165Ho (clone VI-PL2); from Longwood Medical Area Antibody Core (Boston, MA): anti-CD36-150Nd (clone 5-271), anti-CD42b-163Dy (clone HIP1), anti-CD41-149Sm (clone HIP8), anti-CD62P-172Yb (clone AK4), anti-CD63-161Dy (clone H5C6), anti-CD107a-166Er (clone H4A3), anti-CD154-154Sm (clone 24-31); or labeled in-house (described below): anti-CD29-176Yb (Biolegend, San Diego, CA, clone TS2/16), anti-CD42a-155Gd (BD Biosciences, San Jose, CA, clone ALMA.16), anti-GPVI-152Sm (EMD Millipore, Billerica, MA, polyclonal IgG), anti-activated αIIbβ3 (PAC1)-159Tb (BD Biosciences, San Jose, CA, clone PAC1).

Techniques: Staining, Expressing

Journal: Scientific Reports

Article Title: Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia

doi: 10.1038/s41598-018-28211-5

Figure Lengend Snippet: MC reveals novel alterations in the platelet surface expression of antigens in Glanzmann thrombasthenia (GT) patients. Citrate-anticoagulated blood samples from healthy donors (n = 3) and a GT patient (3 separate blood draws from the same patient on 3 different visits) were treated with vehicle, ADP (0.5 or 20 µM) or TRAP (1.5 or 20 µM) for 30 minutes in the presence of a fluorescent-tagged antibody cocktail (A) ( CD41a-PE and CD61-FITC or PAC1-FITC and CD62P-PE) or a custom metal-tagged antibody cocktail ( B ) (CD41-149Sm, CD61-165Ho, PAC1-159Tb, CD62P-172Yb, CD63-161Dy, CD9-171Yb, CD154-154Sm, CD42a-155Gd, CD42b-163Dy, GPVI-152Sm, CD31-145Nd, CD36-150Nd, CD29-176Yb and CD107a-166Er). Samples were fixed in 1% formaldehyde and analyzed by flow cytometry or mass cytometry. Results are expressed as a percentage of the mean fluorescence intensity (MFI; flow cytometry readout) or mean metal intensity (MMI; mass cytometry readout) achieved with 20 µM TRAP in healthy donor platelets (means ± SEM; n = 3). Statistical analysis: 2-way ANOVA was used in conjunction with a Bonferroni post-test to indicate statistical significance; *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: ADP, adenosine diphosphate; FITC, fluorescein isothiocyanate; MC, mass cytometry; PE, phycoerythrin; TRAP, thrombin receptor activating peptide.

Article Snippet: Metal-conjugated monoclonal antibodies were from Fluidigm Corporation (San Francisco, CA): anti-CD9-171Yb (clone SN4 C33A2), anti-CD31-145Nd (clone WM59), anti-CD61-165Ho (clone VI-PL2); from Longwood Medical Area Antibody Core (Boston, MA): anti-CD36-150Nd (clone 5-271), anti-CD42b-163Dy (clone HIP1), anti-CD41-149Sm (clone HIP8), anti-CD62P-172Yb (clone AK4), anti-CD63-161Dy (clone H5C6), anti-CD107a-166Er (clone H4A3), anti-CD154-154Sm (clone 24-31); or labeled in-house (described below): anti-CD29-176Yb (Biolegend, San Diego, CA, clone TS2/16), anti-CD42a-155Gd (BD Biosciences, San Jose, CA, clone ALMA.16), anti-GPVI-152Sm (EMD Millipore, Billerica, MA, polyclonal IgG), anti-activated αIIbβ3 (PAC1)-159Tb (BD Biosciences, San Jose, CA, clone PAC1).

Techniques: Expressing, Flow Cytometry, Mass Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Human iPSC-derived Committed Cardiac Progenitors Generate Cardiac Tissue Grafts in a Swine Ischemic Cardiomyopathy Model without Triggering Ventricular Arrhythmias

doi: 10.1101/2024.02.14.580375

Figure Lengend Snippet: A) Flow cytometry for cell surface markers (CD56, CXCR4, KDR, PDGRFα) and the cardiomyocyte-specific sarcomeric α-actinin (SAA) protein expression directly after thawing the CCPs. B) Seven days after plating CCPs on vitronectin in RPMI+B27 analysis by flow cytometry (left panel) for cardiomyocyte (SAA) and immunofluorescence labeling (right panels) for cardiomyoctye markers Nkx2.5 SAA, cardiac troponin I (cTnI) and cardiac troponin T (cTnT). C) Twelve days after plating CCPs in Vasculife LS1020 media analysis by flow cytometry for endothelial markers CD31 and CD144 and immunolabeling (right panel) for CD31. D) Dissociated day 12 cells in Vasculife LS1020 plated for one day in undliluted Matrigel form tubes.

Article Snippet: Cell surface protein expression was performed by labeling 2×10 cells for KDR-PE (R&D Systems, FAB357P) + PDGFRα-APC (Cell Signaling, 5876S); CD56-APC (BD Biosciences, 555518) + CXCR4-PE (BioLegend, 306506); EpCAM-FITC (Miltenyi, 130-113-263); CD31-FITC (BD Pharmingen, 555445) + CD144-647 (BD Pharmingen, 561567); CD140b-PE (BD Pharmingen, 558821) + CD90-APC (BD Pharmingen, 559869) diluted in wash buffer (DPBS(-/-)+2% FBS) at 4°C for up to 60 minutes followed by washing and analysis with a BD Accuri flow cytometer.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Labeling, Immunolabeling

Journal: bioRxiv

Article Title: Human iPSC-derived Committed Cardiac Progenitors Generate Cardiac Tissue Grafts in a Swine Ischemic Cardiomyopathy Model without Triggering Ventricular Arrhythmias

doi: 10.1101/2024.02.14.580375

Figure Lengend Snippet: A) Epifluorescence image of porcine heart section with human graft identified by Ku80 immunolabeling and cardiomyoctyes by cTnI immunolabeling. B) Light microscopic image of adjacent histological section stained with Mason’s Trichrome. C-E) Immunofluorescence images of human graft in porcine myocardium immunolabeled as indicated identifies human cardiomyocytes. F-I) Immunofluorescence image of another human graft in porcine myocardium labeled with anti-Ku80 and anti-CD31 to identify human endothelial cells in the graft.

Article Snippet: Cell surface protein expression was performed by labeling 2×10 cells for KDR-PE (R&D Systems, FAB357P) + PDGFRα-APC (Cell Signaling, 5876S); CD56-APC (BD Biosciences, 555518) + CXCR4-PE (BioLegend, 306506); EpCAM-FITC (Miltenyi, 130-113-263); CD31-FITC (BD Pharmingen, 555445) + CD144-647 (BD Pharmingen, 561567); CD140b-PE (BD Pharmingen, 558821) + CD90-APC (BD Pharmingen, 559869) diluted in wash buffer (DPBS(-/-)+2% FBS) at 4°C for up to 60 minutes followed by washing and analysis with a BD Accuri flow cytometer.

Techniques: Immunolabeling, Staining, Immunofluorescence, Labeling