Journal:

Article Title: Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

doi: 10.1182/blood-2005-07-2755

Figure Lengend Snippet: EYFPki MKs serve as reporters for the DMS. (A) Electron micrograph of a representative terminally differentiated MK, displaying abundant DMS that fills the cytoplasm except for the cell cortex. (B) MK derived from EYFPki mice, in which one GPIIb locus is replaced by modified (myristoyl-acceptor) EYFP cDNA, showing a fluorescent internal membrane system that corresponds to the morphology and distribution of the DMS. MK preparations centrifuged on cover slips were examined by fluorescence deconvolution microscopy; a central z-section is depicted. (C-E) Micrograph of a typical young EYFPki MK, with limited surface fluorescence; the cell is outlined (E) with CD61 antibody. (F-H) Costaining of advanced EYFPki MKs with Arp3 antibody (G) shows no correspondence between membranous EYFP signal and diffuse Arp3 staining. (I-K) Micrographs of EYFPki MKs in the process of elaborating proplatelets. Proplatelet membranes are contiguous with MK internal membranes, revealing directly that the latter provide the reservoir for proplatelet formation. (E) Peripheral blood platelets in EYFPki mice show surface fluorescence and further support a DMS origin for blood platelets. Scale bars: A, 5 μm; F-H, 10 μm; K, 3 μm; all others, 15 μm.

Article Snippet: MKs treated with drugs, shRNA, or corresponding controls were pelleted at the indicated times and incubated for 20 minutes in 100 μL PBS containing either FITC-conjugated CD41 antibody (clone MWReg30), phycoerythrin-conjugated CD61 antibody (clone 2C9.G2), or the corresponding isotype controls (all from Becton Dickinson, Franklin Lakes, NJ).

Techniques: Derivative Assay, Modification, Membrane, Fluorescence, Microscopy, Staining

Journal:

Article Title: Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

doi: 10.1182/blood-2005-07-2755

Figure Lengend Snippet: The megakaryocyte DMS accumulates PI-4,5-P2 late in differentiation. MKs were retrovirally transduced with EGFP-tagged pleckstrin homology (PH) domain derived from phospholipase C (PLC)δ1, a specific probe for membrane-associated PI-4,5-P2, and examined by fluorescence deconvolution microscopy. (A-D) In young MKs, only the plasma membrane shows PI-4,5-P2 accumulation, as described for other cells. (A) EYFP merged with nuclear DAPI stain, (B) EYFP alone, (C) CD61 stain alone, (D) merger of EYFP and CD61 signals, showing overlap. (E) In MKs of advanced differentiation, internal membranes stain strongly with PH-PLCδ1. The cell-surface signal is weak, and the predominant staining is internal. (F) Continued cytologic maturation, judged by increased cell size and nuclear complexity, results in elaborate staining of internal membranes, which resemble the DMS as seen by electron microscopy and in EYFPki mice, with virtual loss of cell-surface signal (see also Video S1). (G-I) Costaining of PH(PLCδ1)–EGFP–expressing MKs (G) with CD41 antibody (H), a surface and internal marker of MKs. Double staining (I) highlights the cortical absence of PI-4,5-P2. (J-K) Proplatelets demonstrate PI-4,5-P2 staining in contiguity (arrows) with internal membranes. Scale bars: A-D, 10 μm; E, F, J, K, 15 μm; G-I, 20 μm.

Article Snippet: MKs treated with drugs, shRNA, or corresponding controls were pelleted at the indicated times and incubated for 20 minutes in 100 μL PBS containing either FITC-conjugated CD41 antibody (clone MWReg30), phycoerythrin-conjugated CD61 antibody (clone 2C9.G2), or the corresponding isotype controls (all from Becton Dickinson, Franklin Lakes, NJ).

Techniques: Transduction, Derivative Assay, Membrane, Fluorescence, Microscopy, Clinical Proteomics, Staining, Electron Microscopy, Expressing, Marker, Double Staining

Journal:

Article Title: Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

doi: 10.1182/blood-2005-07-2755

Figure Lengend Snippet: Actin fibers assemble near MK internal membranes and are required for proplatelet formation. (A-F) Wild-type MKs infected with PH(PLCδ1)–EGFP retrovirus were fixed, permeabilized, and labeled with Texas Red–phalloidin and DAPI nuclear stain on culture day 4. A central z-section in panel A reveals internal, PI-4,5-P2–containing membranes. Phalloidin-stained actin filaments appear in a reticular network that colocalizes partially with PH(PLCδ1)–EGFP at one cell pole (B), as judged by merger of fluorescent signals (C). PI-4,5-P2–positive proplatelet membranes also associate closely with actin filaments, suggesting continuity of the spatial relationship established in the cell body (D-F). (G-I) MKs were infected with retroviruses to express EGFP-tagged N-WASp CA domain, and infected cells were identified by fluorescence microscopy. The tagged CA domain colocalizes extensively with Texas Red–labeled Arp3, as shown by the merger of fluorescent signals. (J) Phalloidin staining of WASp-CA–expressing cells confirms disruption of actin filaments, which also failed to colocalize with the EGFP-CA fragment (data not shown). (K) EGFP-CA and control retroviruses were used to infect MKs on culture day 2, and proplatelet formation was scored on day 4. Expression of the isolated CA domain substantially reduced proplatelet formation. Results are averaged from 3 independent experiments, and are presented with standard deviations (error bars). (L) WASP-CA–infected MKs are large, have multilobed nuclei, and extend rare proplatelets (inset), findings that argue against general toxicity. (M) Proplatelet formation by cytochalasin D–treated MKs on culture day 4. Results show the effects of introducing cytoD into MK cultures between days 2.5 and 3.5 and are averaged over 3 independent experiments. Ctrl indicates DMSO treatment for 2.5 days. (N) Representative EYFPki MK treated with cytoD on culture day 2, showing the characteristically elaborate internal membrane structure. (O) Cells treated with DMSO (control) or with 0.2 μM cytoD on culture days 2.5 or 3 were assessed for cell size on day 4. The fraction of FSChigh MKs (CD61+ cells) is shown from 2 independent experiments. MKs were reduced slightly in size but showed little difference over the same interval in which proplatelet formation (M) was notably inhibited. Scale bars: A-I, N, 15 μm; J, L, 10 μm.

Article Snippet: MKs treated with drugs, shRNA, or corresponding controls were pelleted at the indicated times and incubated for 20 minutes in 100 μL PBS containing either FITC-conjugated CD41 antibody (clone MWReg30), phycoerythrin-conjugated CD61 antibody (clone 2C9.G2), or the corresponding isotype controls (all from Becton Dickinson, Franklin Lakes, NJ).

Techniques: Infection, Labeling, Staining, Fluorescence, Microscopy, Expressing, Disruption, Control, Isolation, Membrane

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Treatment of β -Thalassemia/Hemoglobin E with Antioxidant Cocktails Results in Decreased Oxidative Stress, Increased Hemoglobin Concentration, and Improvement of the Hypercoagulable State

doi: 10.1155/2015/537954

Figure Lengend Snippet: Procoagulation parameters, markers of platelet activation, and blood coagulation tests in responders.

Article Snippet: To measure the markers of platelet activation, platelets in citrated blood were activated by adenosine diphosphate and then labeled with anti-human CD41a-FITC, anti-CD62-FITC (Cymbus Biotechnology, Chandlers Ford, UK), and anti-PAC1-FITC (Becton, Dickinson and Co., Heidelberg, Germany).

Techniques: Activation Assay, Coagulation, Activity Assay, Expressing

Journal: Lymphatic Research and Biology

Article Title: Lymphatic Pump Treatment Augments Lymphatic Flux of Lymphocytes in Rats

doi: 10.1089/lrb.2010.0009

Figure Lengend Snippet: Lymphatic Pump Treatment Increases Lymphocyte Flux in Cisterna Chyli Lymph

Article Snippet: Two-color immunofluorescent staining was performed to identify specific lymphocyte populations using FITC or PE-labeled goat anti-rat isotype control IgG2b, FITC-anti-rat CD3, PE-anti-rat B cell, PE-anti-rat CD4, PE-anti-rat CD8, FitC-anti-rat CD61 (β7), or PE-anti-rat CD 49 (α4) monoclonal antibodies (mAb) (Serotech, Raleigh, NC).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Mutation in GP1BB Reveals the Role of the Cytoplasmic Domain of GPIbβ in the Pathophysiology of Bernard-Soulier Syndrome and GPIb-IX Complex Assembly

doi: 10.3390/ijms221910190

Figure Lengend Snippet: Flow cytometry analysis of platelet surface glycoproteins.

Article Snippet: FITC-conjugated VIPL2 against glycoprotein GPIIIa (CD61) was from Immunostep (Salamanca, Spain).

Techniques: Flow Cytometry