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  • 99
    Lonza trypsin ethylenediaminetetraacetic acid
    Trypsin Ethylenediaminetetraacetic Acid, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher trypsin edta
    The suppression of OCs on T-cell proliferation is IFN-γ and CD40L dependent Proliferation of CD4 + T cells stimulated by (A) α-CD3/CD28 Dynabeads at a T/Bead ratio of 2:1 or (B) allogeneic DCs at a T/DC ratio of 10:1 in <t>Transwells</t> in the absence or presence of autologous OCs with IFN-γ neutralizing or IFNGR1 blocking antibodies (20 μg/mL) for 4–6 d. (C) Proliferation of CD4 + T cells stimulated by allogeneic DCs under the contact coculture of autologous OCs with IFN-γ neutralizing, CD40 or CD40L blocking antibodies (20 μg/mL). (D) Expression of IFNGR1 and CD40 on OCs, as measured by flow cytometry. OCs coculutred with α-CD3/CD28 Dynabeads-activated CD4 + T cells in Transwell system were harvested by <t>Trypsin/EDTA</t> detachment, stained by antibodies and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.
    Trypsin Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore edta
    Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM <t>EDTA</t> in FGM-2 culture media containing 1% <t>FBS.</t> c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher versene
    L-selectin binds to HIV envelope gp120. a Blank subtracted sensorgrams of serially diluted recombinant gp120 BAL binding to immobilized CD62L-Fc. b Binding of glycosylated and deglycosylated gp120 at ~2 µM concentration to immobilized CD62L-Fc and DCSIGN-Fc. The BIAcore bindings were performed as triplicates using PNGase F treated and untreated gp120s at equal concentrations. Deglycosylated gp120 BAL and gp120 SF33 bound significantly less to both CD62L and DC-SIGN compared to their glycosylated gp120. c ELISA binding of CD62L-Fc to immobilized gp120 in the presence of 10 mM lactose, GlcNAc ( N -acetyl- d -glucosamine), sialyl-lactose, sialyl-Lewis x (sLe x ), fucoidan, heparin, and <t>EDTA.</t> sLe x , fucoidan, and heparin are known ligands of CD62L and inhibited gp120 binding. The p values are calculated with respect to PBS control. d Number of gp120-QDots bound to untransfected or L-selectin-transfected HeLa cells and counted using TIRF microscopy. e Flow cytometry analysis of gp120-QDots binding to PBMC in the presence and absence of anti-CD4 (RPA-T4), and anti-CD62L (DREG-56) antibodies. f Capture of HIV-1 BAL virus (1:1000 dilution) with plate-immobilized 10 µg soluble CD62L in the presence and absence of 5 mM EDTA, CD4, BSA, IgG or blank (PBS control). The captured virus was quantified using p24 ELISA (PerkinElmer). g The binding of HIV-1 BAL virus (1:1000 dilution) to 10 6 CD62L-transfected or untransfected 293T cells. h The binding of HIV-1 BAL virus to 10 6 sorted CD62L + or CD62L − CD8 + T cells. The error bars indicate standard deviations from the means. The p values of all figures are calculated using unpaired Student's t test with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The results included in panels a − d , f − h are from at least two independent experiments with all data included
    Versene, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin edta
    Inhibition of <t>KSHV</t> infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% <t>trypsin-EDTA</t> for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.
    Trypsin Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ethylenediaminetetraacetic acid edta
    Transport of catechin, EGCG, and their niosomes by human intestinal Caco-2 cells. Notes: Schematic diagram of transcellular transport and different transporters in Caco-2 cells ( A ); effect of concentration on the flux of catechin, EGCG, and their niosomes (0, 10, 20, 50, and 100 μg/mL) through Caco-2 monolayer from apical to basolateral chamber ( B ); effect of temperature (4°C and 37°C), ATP inhibitor (sodium azide), P-gp inhibitor (verapamil), MRP2 inhibitor (MK-571) and absorption enhancer <t>(EDTA)</t> on transport of 100 μg/mL different drugs after 6 hours ( C ). Data are presented as mean ± SD (n=3). Abbreviations: EDTA, <t>ethylenediaminetetraacetic</t> acid; EGCG, (−)-epigallocatechin gallate; MRP2, multidrug resistance-associated protein 2; P-gp, permeability glycoprotein; SD, standard deviation.
    Ethylenediaminetetraacetic Acid Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam antigen retrieval
    Transport of catechin, EGCG, and their niosomes by human intestinal Caco-2 cells. Notes: Schematic diagram of transcellular transport and different transporters in Caco-2 cells ( A ); effect of concentration on the flux of catechin, EGCG, and their niosomes (0, 10, 20, 50, and 100 μg/mL) through Caco-2 monolayer from apical to basolateral chamber ( B ); effect of temperature (4°C and 37°C), ATP inhibitor (sodium azide), P-gp inhibitor (verapamil), MRP2 inhibitor (MK-571) and absorption enhancer <t>(EDTA)</t> on transport of 100 μg/mL different drugs after 6 hours ( C ). Data are presented as mean ± SD (n=3). Abbreviations: EDTA, <t>ethylenediaminetetraacetic</t> acid; EGCG, (−)-epigallocatechin gallate; MRP2, multidrug resistance-associated protein 2; P-gp, permeability glycoprotein; SD, standard deviation.
    Antigen Retrieval, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore trypsin edta solution
    Both surface binding and intracellular entry by fusion of HIV-1 virions are enhanced by endosome inhibitors. SupT1 cells were pretreated for 30 min at 37°C with medium (columns 1 and 2), bafilomycin A1 (100 nM; columns 3 and 4), or NH 4 Cl (10 mM; columns 5 and 6). Cells were then incubated with GFP-Vpr-labeled HIV-1 virions (200 ng of p24 Gag) for 3 h at 37°C. Cells were either washed in <t>PBS</t> (columns 1, 3, and 5) or treated with a <t>trypsin-EDTA</t> solution to remove surface-bound virions (columns 2, 4, and 6) followed by flow cytometric analysis of living cells. The percentage of SupT1 cells displaying GFP epifluorescence is indicated on the ordinate. The data presented are from a representative experiment repeated three times with comparable results.
    Trypsin Edta Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 3526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson potassium edta
    Preparation of HDL associated <t>LCAT.</t> Size exclusion chromatography was perfo rmed at ambient temperature using a Superose-6 10/300 column on a Bio-Rad FPLC system (Hercules, CA). A 200 µl aliquot of plasma was injected per run, and eluted in PBS with 1 mM <t>EDTA</t> at a flow rate of 0.2 ml/min. Total cholesterol in each fraction was measured using the Cholesterol E Kit. LCAT activity was determined as following: A. 20 µl of each fraction of human plasma were incubated at 37ºC for 5 h; B. 2.5 µl of each fraction of WT mouse plasma were incubated at 37ºC for 2 h; C. 10 µl of each fraction of apoE-null mouse plasma were incubated at 37ºC for 4 h.
    Potassium Edta, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad edta
    IFT27 directly interacts with nucleotide-empty <t>ARL6</t> (A) LAP eluates from IFT27 LAP and LAP IFT88 were analyzed by immunoblotting for IFT-B subunits (IFT88, IFT57), IFT27 and ARL6. Twice as much of the LAP IFT88 eluate was loaded compared to the IFT27 LAP eluate. Note that the IFT27 antibody preferentially recognizes murine IFT27 over human IFT27, accounting for the lower signal intensity of IFT27 S-tag in the IFT27 LAP lane compared to that of murine IFT27 in the LAP IFT88 lane. (B) Co-transfections/co-immunoprecipitations were performed with all combinations of “GTP-locked” or “GDP-locked” variants of ARL6 and IFT27. IFT25 was co-transfected with IFT27 LAP to ensure IFT27 stability. (C) IFT25/IFT27 LAP -decorated beads were used to capture overexpressed Myc ARL6 out of HEK cell lysate in the presence or absence of <t>EDTA.</t> (D) The IFT25/IFT27-GST complex, GST, ARL6 and SAR1A were expressed in bacteria and purified to near-homogeneity before SDS-PAGE and Coomassie staining (left panel). IFT25/IFT27-GST was mixed with ARL6 or SAR1A in the presence of various nucleotides and complexes were recovered on Glutathione Sepharose beads before LDS elution, SDS-PAGE and Coomassie staining (middle panel). In a similar experiment, IFT25/IFT27-GST was mixed with ARL6 in the presence of GTPγS, EDTA or GDP/AlF 4 − (right panel). (E) ARL6, either alone or mixed with the IFT25/IFT27 complex and EDTA was resolved by size exclusion chromatography (Superdex 200). Size markers: 66.9 kDa (Thyroglobulin), 35.0 kDa (β-lactoglobulin) and 6.5 kDa (Aprotinin). .
    Edta, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson edta tubes
    Study 3: Comparison of PBMC isolation methods and SMN measures from various blood fractions. Due to the variability in SMN signal and protein concentrations seen with <t>CPT</t> tube PBMC isolation, other methods were explored using the four subjects from Study 2. A : PBMC yield was greatest in samples collected with <t>EDTA</t> tubes and subsequent Lymphoprep gradient separation, and showed no statistically significant changes with isolation delays of up to 24 h. B : Total soluble protein tended to increase with 24 h delays in samples collected by EDTA tube and isolated by CPT tubes, while there was no obvious change in protein concentrations in EDTA/Lymphoprep processing. C : SMN as measured by total protein tended to decrease with isolation delays with EDTA/CPT processing. SMN signals were similar with delays up to 24 h with EDTA/Lymphoprep processing. D : SMN by PBMC counts was variable for both EDTA/CPT and EDTA/Lymphoprep processing methods. However, the EDTA/Lymphoprep values were generally overlapping and did not appear to decrease from the no-delay timepoint (t = 0). In Figure 3 body of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median.
    Edta Tubes, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher tbe buffer
    Characterization of barcoded 601 (BC-601) DNA a , BC-601 DNA prepared for all 115 nucleosome library members as described in Methods (Barcoded 601 (BC-601) DNA preparation). Ligation products are 192 bp in size and were visualized by polyacrylamide gel electrophoresis (5% acrylamide, <t>0.5×</t> <t>TBE,</t> 200 V, 40 min) and staining with SYBR Safe DNA gel stain. A faint band corresponding to unligated 601 DNA (601) is slightly visible in certain cases. b .
    Tbe Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher trypsin ethylenediaminetetraacetic acid edta
    In vitro characterization of neuronal cell sheet derived from human induced pluripotent stem cells (hiPSCs). (A) A schematic representation of neuronal sheet formation from hiPSCs and transplantation. Embryoid bodies were developed from undifferentiated hiPSCs in a floating condition for 4 d. Then the cells were cultured in fibronectin-coated dishes for 4 d during which retinoic acid (RA), noggin (NOG), and cyclopamine (CyP; 3 factors) were introduced twice (on days 5 and 7). In some experiments, sonic hedgehog (SHH) were introduced instead of adding CyP, together with RA and NOG in order to compare the effects of CyP with those of SHH. Thereafter, the cells cultured with RA, NOG, and CyP were harvested on day 8. An aliquot of the cells was cultured in 24-well culture plates for 12 to 16 d and then harvested using <t>trypsin/ethylenediaminetetraacetic</t> acid <t>(EDTA)</t> solution for transplantation. The remaining cells were cultured on temperature-responsive gelation polymer-coated plate (temp. resp. polymer) for 12 to 16 d depending on the neuronal maturation, where they extended axon-like processes, leading to the formation of neuronal cell sheet. The neuronal cell sheet was recovered by lowering the temperature of culture plate below 22°C. Schedule for brain injury, transplantation, and motor function test was reported previously. 10 , 20 , 28 , 29 The cells on day 8 were used as neural stem/progenitor cells for comparison. (B) Stereomicroscopic view of the neuronal cell sheet cultured in a 48-well (diameter 12 mm) culture plate. Right lower part of the sheet detached from the bottom of the plate. (C) Stereomicroscopic view of the sheet in a 10-cm culture dish (diameter 90 mm). (D, E) Hematoxylin and eosin staining of the neuronal cell sheet (D, lower; E, higher magnification). Flow cytometric analysis of the cells in the sheet treated with trypsin/EDTA revealed that more than 75% of the cells expressed human NCAM (data not shown). (F, G) The neuronal cell sheet at day 19 was stained with anti-human nuclei (F) and anti-β-tubulin antibodies (G). (H) Real-time polymerase chain reaction analyses of the Figure 1. (continued) . neuronal cell sheet. For comparison, neural cells on day 8 (neural cells were cultured in the same manner except that SHH was added instead of CyP and were harvested on day 8 before making cell sheet) were included. The cell sheet expressed Foxp2, Fezf2, Igfbp4, CTIP2, and synaptophysin mRNA predominantly. CRIM1 mRNA was expressed throughout the culture period. Thus, the cell sheet expressed mRNAs of cortical motoneuron–associated proteins. A white horizontal bar represents 200 μm in panel D, 100 μm in panels E and F, and 50 μm in panel G. UiPSCs, undifferentiated-induced pluripotent stem cells; CRIM1, cysteine-rich motor neuron 1 protein precursor; MEF, mouse embryonic fibroblasts; floating indicates floating condition; Foxp2, forkhead box p2; Fezf2, forebrain embryonic zinc finger family zinc finger 2; Igfbp4, insulin-like growth factor-binding protein 4; CTIP2, COUP-TF-interacting protein 2; mRNA, messenger RNA.
    Trypsin Ethylenediaminetetraacetic Acid Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson trypsin edta
    Ecto-tagged β1 integrins bind soluble ligand, restore surface levels of endogenous α5 integrins, and display normal activation indices. a Flow cytometry histograms showing binding of soluble <t>FN9-10</t> to parental and a subset of reconstituted KO fibroblasts, in native conditions (filled orange peak ), <t>EDTA-inhibited</t> conditions ( blue peak ), or Mn 2 + -treated conditions ( red peak ). Full data set in Supplementary Fig. 3a . b , c Quantification of surface levels of human β1 ( b ) and mouse α5 integrins ( c ) measured by flow cytometry on parental and reconstituted β1 integrin fl/fl and KO fibroblasts. d Activation index of surface α5β1 integrins on parental and reconstituted β1 integrin fl/fl and KO fibroblasts, calculated as (FN9-10 binding in native conditions—FN9-10 binding in EDTA-inhibited conditions)/surface levels of α5 integrins. All data b – d is shown as mean ± SEM from four independent experiments. Statistical analysis was performed using one-way repeated measures ANOVA with Dunnett post hoc test. Each column was compared to KO no-tag β1 and * p
    Trypsin Edta, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson edta vacutainer tubes
    Kinetic analysis of circulating lymphocytes following influenza virus infection. Three ferrets each were inoculated i.n. with 10 6 PFU of virus. Blood was collected on days 3 (A), 7 (B), and 14 to 16 (C) p.i. in <t>EDTA</t> <t>Vacutainer</t> tubes and analyzed with
    Edta Vacutainer Tubes, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 790 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific edta
    GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by <t>EDTA,</t> confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in <t>PBS</t> (pH 3.5).
    Edta, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris edta buffer
    Ethanol Precipitation Dissociates the Catalytic Subunit from the Regulatory Subunits of PP2A. The effect of ethanol precipitation on the molecular form of PP2A was determined following size exclusion chromatography in <t>Tris–EDTA</t> buffer containing 10 mM β -mercaptoethanol and 100 mM NaCl on a Sephacryl 300HR column of the crude S100 fraction (3.0 mg protein) and S100 fraction following ethanol precipitation, extraction, and ammonium sulfate precipitation to form the EtOH-AS65 fraction (0.15 mg protein). PP2Ac in the fractions was determined by measuring the phosphatase activity toward a phosphothreonine peptide. Phosphatase activity is reported as nmol Pi produced during a 10 min incubation period per 225 μL of the column fractions. γ -Globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) were employed as molecular weight markers
    Tris Edta Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tae buffer
    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the <t>deproteinized</t> reaction products were separated by 1% agarose gel electrophoresis in 1× <t>TAE</t> buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.
    Tae Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2058 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson k2 edta
    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the <t>deproteinized</t> reaction products were separated by 1% agarose gel electrophoresis in 1× <t>TAE</t> buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.
    K2 Edta, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences trypsin edta
    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the <t>deproteinized</t> reaction products were separated by 1% agarose gel electrophoresis in 1× <t>TAE</t> buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.
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    Millipore edta free protease inhibitor cocktail
    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the <t>deproteinized</t> reaction products were separated by 1% agarose gel electrophoresis in 1× <t>TAE</t> buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.
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    Mediatech trypsin edta
    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the <t>deproteinized</t> reaction products were separated by 1% agarose gel electrophoresis in 1× <t>TAE</t> buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.
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    Image Search Results


    The suppression of OCs on T-cell proliferation is IFN-γ and CD40L dependent Proliferation of CD4 + T cells stimulated by (A) α-CD3/CD28 Dynabeads at a T/Bead ratio of 2:1 or (B) allogeneic DCs at a T/DC ratio of 10:1 in Transwells in the absence or presence of autologous OCs with IFN-γ neutralizing or IFNGR1 blocking antibodies (20 μg/mL) for 4–6 d. (C) Proliferation of CD4 + T cells stimulated by allogeneic DCs under the contact coculture of autologous OCs with IFN-γ neutralizing, CD40 or CD40L blocking antibodies (20 μg/mL). (D) Expression of IFNGR1 and CD40 on OCs, as measured by flow cytometry. OCs coculutred with α-CD3/CD28 Dynabeads-activated CD4 + T cells in Transwell system were harvested by Trypsin/EDTA detachment, stained by antibodies and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Human Osteoclasts are Inducible Immunosuppressive Cells in Response to T cell-Derived IFN-γ and CD40 Ligand in vitro

    doi: 10.1002/jbmr.2294

    Figure Lengend Snippet: The suppression of OCs on T-cell proliferation is IFN-γ and CD40L dependent Proliferation of CD4 + T cells stimulated by (A) α-CD3/CD28 Dynabeads at a T/Bead ratio of 2:1 or (B) allogeneic DCs at a T/DC ratio of 10:1 in Transwells in the absence or presence of autologous OCs with IFN-γ neutralizing or IFNGR1 blocking antibodies (20 μg/mL) for 4–6 d. (C) Proliferation of CD4 + T cells stimulated by allogeneic DCs under the contact coculture of autologous OCs with IFN-γ neutralizing, CD40 or CD40L blocking antibodies (20 μg/mL). (D) Expression of IFNGR1 and CD40 on OCs, as measured by flow cytometry. OCs coculutred with α-CD3/CD28 Dynabeads-activated CD4 + T cells in Transwell system were harvested by Trypsin/EDTA detachment, stained by antibodies and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.

    Article Snippet: To detect the viability of OCs during the coculture with α-CD3/CD28 DynaBeads-stimulated T cells in Transwells, coculutred OCs were digested from the culture plate with Trypsin/EDTA, and stained by using LIVE/DEAD® Fixable Dead Cell Stain Kit (Molecular Probes, Life Technologies).

    Techniques: Blocking Assay, Expressing, Flow Cytometry, Cytometry, Staining

    Sevoflurane and rat brain endothelial cell size in H/R injury. RBE4 cells were exposed to severe hypoxia (0.2% oxygen) for 24 hours, followed by a 4-hour period of reoxygenation in air (H/R+air) or air enriched with sevoflurane (H/R+sevo). Cells in the normoxia group remained in a normal cell culture environment with 21% oxygen for the full 28 hours. After detachment with trypsin-EDTA and viability staining, cells were analyzed with the flow cytometer or image stream, respectively. FSC-A as an indirect measure of cell size was assessed in conventional flow cytometry ( A ), while the cell size of each cell was assessed with the image stream method ( B ). The histogram shows the distribution pattern of FSC-A seen in the flow cytometry. n = 5 independent experiments, with 500 cells analyzed in each condition. The bar graph shows mean values and standard deviations. n = 4 Image stream X experiments, at least 20,000 cells analyzed in each condition. Analysis with one way ANOVA and Bonferroni correction. *** p

    Journal: PLoS ONE

    Article Title: Sevoflurane protects rat brain endothelial barrier structure and function after hypoxia-reoxygenation injury

    doi: 10.1371/journal.pone.0184973

    Figure Lengend Snippet: Sevoflurane and rat brain endothelial cell size in H/R injury. RBE4 cells were exposed to severe hypoxia (0.2% oxygen) for 24 hours, followed by a 4-hour period of reoxygenation in air (H/R+air) or air enriched with sevoflurane (H/R+sevo). Cells in the normoxia group remained in a normal cell culture environment with 21% oxygen for the full 28 hours. After detachment with trypsin-EDTA and viability staining, cells were analyzed with the flow cytometer or image stream, respectively. FSC-A as an indirect measure of cell size was assessed in conventional flow cytometry ( A ), while the cell size of each cell was assessed with the image stream method ( B ). The histogram shows the distribution pattern of FSC-A seen in the flow cytometry. n = 5 independent experiments, with 500 cells analyzed in each condition. The bar graph shows mean values and standard deviations. n = 4 Image stream X experiments, at least 20,000 cells analyzed in each condition. Analysis with one way ANOVA and Bonferroni correction. *** p

    Article Snippet: Flow cytometry and cell size measurement For flow and imaging cytometry cells were washed twice with 2.5mmol EDTA in PBS, detached with trypsin-EDTA solution (0.05%) (Thermo, Reinach, Switzerland) and put through a 35μm cell strainer (Falcon, Corning, USA) after resuspension.

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

    Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Article Snippet: Cell adhesion assays were performed using human lung fibroblasts (Lonza) in FGM-2 medium (Lonza) or human umbilical vein endothelial cells (HUVEC; Lonza) in EGM-2 medium (Lonza) supplemented with 1% fetal bovine serum (FBS) and 100 µg/mL VEGF-A165, with or without 5 mM EDTA (Sigma-Aldrich).

    Techniques: In Vitro, Cell Culture, Incubation, CyQUANT Assay

    L-selectin binds to HIV envelope gp120. a Blank subtracted sensorgrams of serially diluted recombinant gp120 BAL binding to immobilized CD62L-Fc. b Binding of glycosylated and deglycosylated gp120 at ~2 µM concentration to immobilized CD62L-Fc and DCSIGN-Fc. The BIAcore bindings were performed as triplicates using PNGase F treated and untreated gp120s at equal concentrations. Deglycosylated gp120 BAL and gp120 SF33 bound significantly less to both CD62L and DC-SIGN compared to their glycosylated gp120. c ELISA binding of CD62L-Fc to immobilized gp120 in the presence of 10 mM lactose, GlcNAc ( N -acetyl- d -glucosamine), sialyl-lactose, sialyl-Lewis x (sLe x ), fucoidan, heparin, and EDTA. sLe x , fucoidan, and heparin are known ligands of CD62L and inhibited gp120 binding. The p values are calculated with respect to PBS control. d Number of gp120-QDots bound to untransfected or L-selectin-transfected HeLa cells and counted using TIRF microscopy. e Flow cytometry analysis of gp120-QDots binding to PBMC in the presence and absence of anti-CD4 (RPA-T4), and anti-CD62L (DREG-56) antibodies. f Capture of HIV-1 BAL virus (1:1000 dilution) with plate-immobilized 10 µg soluble CD62L in the presence and absence of 5 mM EDTA, CD4, BSA, IgG or blank (PBS control). The captured virus was quantified using p24 ELISA (PerkinElmer). g The binding of HIV-1 BAL virus (1:1000 dilution) to 10 6 CD62L-transfected or untransfected 293T cells. h The binding of HIV-1 BAL virus to 10 6 sorted CD62L + or CD62L − CD8 + T cells. The error bars indicate standard deviations from the means. The p values of all figures are calculated using unpaired Student's t test with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The results included in panels a − d , f − h are from at least two independent experiments with all data included

    Journal: Nature Communications

    Article Title: HIV-1 targets L-selectin for adhesion and induces its shedding for viral release

    doi: 10.1038/s41467-018-05197-2

    Figure Lengend Snippet: L-selectin binds to HIV envelope gp120. a Blank subtracted sensorgrams of serially diluted recombinant gp120 BAL binding to immobilized CD62L-Fc. b Binding of glycosylated and deglycosylated gp120 at ~2 µM concentration to immobilized CD62L-Fc and DCSIGN-Fc. The BIAcore bindings were performed as triplicates using PNGase F treated and untreated gp120s at equal concentrations. Deglycosylated gp120 BAL and gp120 SF33 bound significantly less to both CD62L and DC-SIGN compared to their glycosylated gp120. c ELISA binding of CD62L-Fc to immobilized gp120 in the presence of 10 mM lactose, GlcNAc ( N -acetyl- d -glucosamine), sialyl-lactose, sialyl-Lewis x (sLe x ), fucoidan, heparin, and EDTA. sLe x , fucoidan, and heparin are known ligands of CD62L and inhibited gp120 binding. The p values are calculated with respect to PBS control. d Number of gp120-QDots bound to untransfected or L-selectin-transfected HeLa cells and counted using TIRF microscopy. e Flow cytometry analysis of gp120-QDots binding to PBMC in the presence and absence of anti-CD4 (RPA-T4), and anti-CD62L (DREG-56) antibodies. f Capture of HIV-1 BAL virus (1:1000 dilution) with plate-immobilized 10 µg soluble CD62L in the presence and absence of 5 mM EDTA, CD4, BSA, IgG or blank (PBS control). The captured virus was quantified using p24 ELISA (PerkinElmer). g The binding of HIV-1 BAL virus (1:1000 dilution) to 10 6 CD62L-transfected or untransfected 293T cells. h The binding of HIV-1 BAL virus to 10 6 sorted CD62L + or CD62L − CD8 + T cells. The error bars indicate standard deviations from the means. The p values of all figures are calculated using unpaired Student's t test with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The results included in panels a − d , f − h are from at least two independent experiments with all data included

    Article Snippet: RPMI, penicillin/streptomycin, fetal bovine serum (FBS), HEPES, and Versene were purchased from Invitrogen Corporation (Carlsbad, CA).

    Techniques: Recombinant, Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Microscopy, Flow Cytometry, Cytometry, Recombinase Polymerase Amplification

    L-selectin facilitates HIV-1 infection of CD4 + T cells. a HIV infection of CD62L-transfected clones (CD62L-CEM #2 and #25) or untransfected Rev-CEM cells, as well as CD62L knockdown clones, CD62L KD #3, #8, and #32. CD62L KD #8 and #32 lost CD62L expression whereas #3 retained parental CD62L expression (Supplementary Figure 4 ). b HIV infection of CEM cell lines in the presence and absence of 1 µg/mL polybrene. c HIV-1 BAL infection of PBMC in the presence of EDTA or anti-CD4 (RPA-T4). The infection was measured by intracellular p24 + staining. d Dose-dependent HIV-1 BAL infection of PBMC in the presence of CD62L blocking antibody DREG-56 or isotype controls. Infections were measured as copy numbers of viral DNA by real-time PCR and displayed as % relative to the isotype controls. The results are from at least two independent experiments and statistics are performed without data rejections

    Journal: Nature Communications

    Article Title: HIV-1 targets L-selectin for adhesion and induces its shedding for viral release

    doi: 10.1038/s41467-018-05197-2

    Figure Lengend Snippet: L-selectin facilitates HIV-1 infection of CD4 + T cells. a HIV infection of CD62L-transfected clones (CD62L-CEM #2 and #25) or untransfected Rev-CEM cells, as well as CD62L knockdown clones, CD62L KD #3, #8, and #32. CD62L KD #8 and #32 lost CD62L expression whereas #3 retained parental CD62L expression (Supplementary Figure 4 ). b HIV infection of CEM cell lines in the presence and absence of 1 µg/mL polybrene. c HIV-1 BAL infection of PBMC in the presence of EDTA or anti-CD4 (RPA-T4). The infection was measured by intracellular p24 + staining. d Dose-dependent HIV-1 BAL infection of PBMC in the presence of CD62L blocking antibody DREG-56 or isotype controls. Infections were measured as copy numbers of viral DNA by real-time PCR and displayed as % relative to the isotype controls. The results are from at least two independent experiments and statistics are performed without data rejections

    Article Snippet: RPMI, penicillin/streptomycin, fetal bovine serum (FBS), HEPES, and Versene were purchased from Invitrogen Corporation (Carlsbad, CA).

    Techniques: Infection, Transfection, Clone Assay, Expressing, Recombinase Polymerase Amplification, Staining, Blocking Assay, Real-time Polymerase Chain Reaction

    TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P

    Journal: Journal of Cell Science

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

    doi: 10.1242/jcs.217034

    Figure Lengend Snippet: TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P

    Article Snippet: Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265).

    Techniques: Knock-Out, Incubation, Western Blot, Expressing, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Transfection

    Cinnamycin facilitates annexin translocation across membranes in cells. (A) Cinnamycin lipid movement activity. HeLa cells were treated with 1 µM cinnamycin for 50 min at 37°C. Then, recombinant annexin-A5–Cy5 (as a probe for PS) and propidium iodide (PI) (to exclude PI-containing dead cells) were added, and cells were incubated for a further 10 min at 37°C. Annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of annexin-A5–Cy5 binding to live cells are shown ( n =3). (B) Western blotting analysis of cell lysates and eluates of HeLa cells treated with cinnamycin (30 min at 37°C) and then with EDTA (10 min at 37°C) as indicated. Quantification of cell surface annexin A2 and annexin A5 {fold change measured as band intensity [cinnamycin(eluate/lysate)/DMSO(eluate/lysate)]} is shown. Results are mean±s.e.m. from n =3 biological replicates; * P

    Journal: Journal of Cell Science

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

    doi: 10.1242/jcs.217034

    Figure Lengend Snippet: Cinnamycin facilitates annexin translocation across membranes in cells. (A) Cinnamycin lipid movement activity. HeLa cells were treated with 1 µM cinnamycin for 50 min at 37°C. Then, recombinant annexin-A5–Cy5 (as a probe for PS) and propidium iodide (PI) (to exclude PI-containing dead cells) were added, and cells were incubated for a further 10 min at 37°C. Annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of annexin-A5–Cy5 binding to live cells are shown ( n =3). (B) Western blotting analysis of cell lysates and eluates of HeLa cells treated with cinnamycin (30 min at 37°C) and then with EDTA (10 min at 37°C) as indicated. Quantification of cell surface annexin A2 and annexin A5 {fold change measured as band intensity [cinnamycin(eluate/lysate)/DMSO(eluate/lysate)]} is shown. Results are mean±s.e.m. from n =3 biological replicates; * P

    Article Snippet: Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265).

    Techniques: Translocation Assay, Activity Assay, Recombinant, Incubation, Binding Assay, Flow Cytometry, Cytometry, Western Blot

    Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

    doi: 10.1128/JVI.01146-08

    Figure Lengend Snippet: Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.

    Article Snippet: Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ).

    Techniques: Inhibition, Infection, Incubation, Fluorescence, Microscopy, Standard Deviation, Binding Assay, Concentration Assay, Labeling, Purification, Isolation, Polymerase Chain Reaction

    ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

    doi: 10.1128/JVI.01146-08

    Figure Lengend Snippet: ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.

    Article Snippet: Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ).

    Techniques: Binding Assay, Incubation, Concentration Assay, Labeling, Radioactivity, Inhibition, Standard Deviation, Infection, Amplification, Polymerase Chain Reaction, Generated, Clone Assay

    Transport of catechin, EGCG, and their niosomes by human intestinal Caco-2 cells. Notes: Schematic diagram of transcellular transport and different transporters in Caco-2 cells ( A ); effect of concentration on the flux of catechin, EGCG, and their niosomes (0, 10, 20, 50, and 100 μg/mL) through Caco-2 monolayer from apical to basolateral chamber ( B ); effect of temperature (4°C and 37°C), ATP inhibitor (sodium azide), P-gp inhibitor (verapamil), MRP2 inhibitor (MK-571) and absorption enhancer (EDTA) on transport of 100 μg/mL different drugs after 6 hours ( C ). Data are presented as mean ± SD (n=3). Abbreviations: EDTA, ethylenediaminetetraacetic acid; EGCG, (−)-epigallocatechin gallate; MRP2, multidrug resistance-associated protein 2; P-gp, permeability glycoprotein; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Enhanced uptake and transport of (+)-catechin and (-)-epigallocatechin gallate in niosomal formulation by human intestinal Caco-2 cells

    doi: 10.2147/IJN.S59331

    Figure Lengend Snippet: Transport of catechin, EGCG, and their niosomes by human intestinal Caco-2 cells. Notes: Schematic diagram of transcellular transport and different transporters in Caco-2 cells ( A ); effect of concentration on the flux of catechin, EGCG, and their niosomes (0, 10, 20, 50, and 100 μg/mL) through Caco-2 monolayer from apical to basolateral chamber ( B ); effect of temperature (4°C and 37°C), ATP inhibitor (sodium azide), P-gp inhibitor (verapamil), MRP2 inhibitor (MK-571) and absorption enhancer (EDTA) on transport of 100 μg/mL different drugs after 6 hours ( C ). Data are presented as mean ± SD (n=3). Abbreviations: EDTA, ethylenediaminetetraacetic acid; EGCG, (−)-epigallocatechin gallate; MRP2, multidrug resistance-associated protein 2; P-gp, permeability glycoprotein; SD, standard deviation.

    Article Snippet: Chemicals and reagents Catechin, EGCG, sorbitan monostearate (Span 60), CH, dihexadecyl phosphate, fluorescein isothiocyanate (FITC), sulforhodamine B (SRB), fluorescein sodium salt, ascorbic acid (AA), sodium azide, verapamil, 5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid sodium salt hydrate (MK-571), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Concentration Assay, Permeability, Standard Deviation

    Both surface binding and intracellular entry by fusion of HIV-1 virions are enhanced by endosome inhibitors. SupT1 cells were pretreated for 30 min at 37°C with medium (columns 1 and 2), bafilomycin A1 (100 nM; columns 3 and 4), or NH 4 Cl (10 mM; columns 5 and 6). Cells were then incubated with GFP-Vpr-labeled HIV-1 virions (200 ng of p24 Gag) for 3 h at 37°C. Cells were either washed in PBS (columns 1, 3, and 5) or treated with a trypsin-EDTA solution to remove surface-bound virions (columns 2, 4, and 6) followed by flow cytometric analysis of living cells. The percentage of SupT1 cells displaying GFP epifluorescence is indicated on the ordinate. The data presented are from a representative experiment repeated three times with comparable results.

    Journal: Journal of Virology

    Article Title: Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

    doi: 10.1128/JVI.78.3.1375-1383.2004

    Figure Lengend Snippet: Both surface binding and intracellular entry by fusion of HIV-1 virions are enhanced by endosome inhibitors. SupT1 cells were pretreated for 30 min at 37°C with medium (columns 1 and 2), bafilomycin A1 (100 nM; columns 3 and 4), or NH 4 Cl (10 mM; columns 5 and 6). Cells were then incubated with GFP-Vpr-labeled HIV-1 virions (200 ng of p24 Gag) for 3 h at 37°C. Cells were either washed in PBS (columns 1, 3, and 5) or treated with a trypsin-EDTA solution to remove surface-bound virions (columns 2, 4, and 6) followed by flow cytometric analysis of living cells. The percentage of SupT1 cells displaying GFP epifluorescence is indicated on the ordinate. The data presented are from a representative experiment repeated three times with comparable results.

    Article Snippet: Cells were washed with phosphate-buffered saline (PBS) or treated with a trypsin-EDTA solution (1×; Sigma) for 2 min at room temperature to remove surface-bound virions, followed by additional washing and fixation in 1% paraformaldehyde-PBS solution.

    Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry

    Flow cytometric analysis of PHA-activated human lymphoblasts infected with GFP-Vpr-labeled HIV-1 virions. (A) PBMCs activated with PHA and cultured in interleukin-2 for 4 days were inoculated with GFP-Vpr-labeled X4-tropic HIV-1 (300 ng of p24 Gag) at 37 or 4°C as indicated. After 3 h, the cells were washed at room temperature with PBS (−T) or a trypsin-EDTA solution (+T) to remove surface-bound virions. Live cells were then analyzed by flow cytometry to detect GFP epifluorescence. Error bars indicate standard deviations of the mean derived from two independent experiments. (B and C) PHA-activated human lymphoblasts were pretreated for 30 min at 37°C with either medium (panels 2 and 4) or AMD3100 (panels 3 and 5). Cells were then incubated at 37°C for 3 or 24 h with GFP-Vpr-labeled X4-tropic HIV-1 or HIV virions pseudotyped with the VSV-G envelope (200 ng of p24 Gag) at 37°C. Cells were subsequently stained with APC-conjugated CD4 antibodies (B) or PE-conjugated CD4 antibodies (C). Cells were analyzed for GFP epifluorescence and APC or PE immunofluorescence. The percentage of cells in each quadrant is indicated. Data shown are from a representative experiment performed three times with comparable results. Note preferential entry of HIV into CD4 cells and the absence of inhibitory effects of AMD3100 measured either at 3 or 24 h.

    Journal: Journal of Virology

    Article Title: Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

    doi: 10.1128/JVI.78.3.1375-1383.2004

    Figure Lengend Snippet: Flow cytometric analysis of PHA-activated human lymphoblasts infected with GFP-Vpr-labeled HIV-1 virions. (A) PBMCs activated with PHA and cultured in interleukin-2 for 4 days were inoculated with GFP-Vpr-labeled X4-tropic HIV-1 (300 ng of p24 Gag) at 37 or 4°C as indicated. After 3 h, the cells were washed at room temperature with PBS (−T) or a trypsin-EDTA solution (+T) to remove surface-bound virions. Live cells were then analyzed by flow cytometry to detect GFP epifluorescence. Error bars indicate standard deviations of the mean derived from two independent experiments. (B and C) PHA-activated human lymphoblasts were pretreated for 30 min at 37°C with either medium (panels 2 and 4) or AMD3100 (panels 3 and 5). Cells were then incubated at 37°C for 3 or 24 h with GFP-Vpr-labeled X4-tropic HIV-1 or HIV virions pseudotyped with the VSV-G envelope (200 ng of p24 Gag) at 37°C. Cells were subsequently stained with APC-conjugated CD4 antibodies (B) or PE-conjugated CD4 antibodies (C). Cells were analyzed for GFP epifluorescence and APC or PE immunofluorescence. The percentage of cells in each quadrant is indicated. Data shown are from a representative experiment performed three times with comparable results. Note preferential entry of HIV into CD4 cells and the absence of inhibitory effects of AMD3100 measured either at 3 or 24 h.

    Article Snippet: Cells were washed with phosphate-buffered saline (PBS) or treated with a trypsin-EDTA solution (1×; Sigma) for 2 min at room temperature to remove surface-bound virions, followed by additional washing and fixation in 1% paraformaldehyde-PBS solution.

    Techniques: Flow Cytometry, Infection, Labeling, Cell Culture, Cytometry, Derivative Assay, Incubation, Staining, Immunofluorescence

    Flow cytometric analysis of human SupT1 cells incubated with GFP-Vpr-labeled X4-tropic (A) or R5-tropic (B) HIV-1 virions in the presence of medium (panels 2 and 3), neutralizing anti-CD4 antibodies (panels 4 and 5), or AMD3100 (panels 6 and 7). SupT1 T cells were preincubated in the presence or absence of anti-CD4 antibodies or AMD3100 for 30 min at 37°C. Cells were then inoculated with GFP-Vpr-labeled X4-tropic HIV-1 or CCR5-tropic HIV-1 R5 (200 ng of p24 Gag) for 3 h at 37°C. The cells were subsequently washed with PBS (panels 1, 2, 4, and 6) or treated with a trypsin-EDTA solution (panels 3, 5, and 7) to remove surface-bound virions. Live cells were then analyzed by flow cytometry for GFP epifluorescence. The vertical bar indicates a gate established by analysis of uninfected cells (panels 1). The percentage of GFP-positive cells is indicated in the upper right hand corner of each panel. The anti-CD4 antibodies significantly inhibited the entry of both X4-tropic and R5-tropic virions, whereas AMD3100 unexpectedly failed to reduce the overall entry of X4-tropic virions.

    Journal: Journal of Virology

    Article Title: Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

    doi: 10.1128/JVI.78.3.1375-1383.2004

    Figure Lengend Snippet: Flow cytometric analysis of human SupT1 cells incubated with GFP-Vpr-labeled X4-tropic (A) or R5-tropic (B) HIV-1 virions in the presence of medium (panels 2 and 3), neutralizing anti-CD4 antibodies (panels 4 and 5), or AMD3100 (panels 6 and 7). SupT1 T cells were preincubated in the presence or absence of anti-CD4 antibodies or AMD3100 for 30 min at 37°C. Cells were then inoculated with GFP-Vpr-labeled X4-tropic HIV-1 or CCR5-tropic HIV-1 R5 (200 ng of p24 Gag) for 3 h at 37°C. The cells were subsequently washed with PBS (panels 1, 2, 4, and 6) or treated with a trypsin-EDTA solution (panels 3, 5, and 7) to remove surface-bound virions. Live cells were then analyzed by flow cytometry for GFP epifluorescence. The vertical bar indicates a gate established by analysis of uninfected cells (panels 1). The percentage of GFP-positive cells is indicated in the upper right hand corner of each panel. The anti-CD4 antibodies significantly inhibited the entry of both X4-tropic and R5-tropic virions, whereas AMD3100 unexpectedly failed to reduce the overall entry of X4-tropic virions.

    Article Snippet: Cells were washed with phosphate-buffered saline (PBS) or treated with a trypsin-EDTA solution (1×; Sigma) for 2 min at room temperature to remove surface-bound virions, followed by additional washing and fixation in 1% paraformaldehyde-PBS solution.

    Techniques: Flow Cytometry, Incubation, Labeling, Cytometry

    Preparation of HDL associated LCAT. Size exclusion chromatography was perfo rmed at ambient temperature using a Superose-6 10/300 column on a Bio-Rad FPLC system (Hercules, CA). A 200 µl aliquot of plasma was injected per run, and eluted in PBS with 1 mM EDTA at a flow rate of 0.2 ml/min. Total cholesterol in each fraction was measured using the Cholesterol E Kit. LCAT activity was determined as following: A. 20 µl of each fraction of human plasma were incubated at 37ºC for 5 h; B. 2.5 µl of each fraction of WT mouse plasma were incubated at 37ºC for 2 h; C. 10 µl of each fraction of apoE-null mouse plasma were incubated at 37ºC for 4 h.

    Journal: International Journal of Biological Sciences

    Article Title: An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

    doi:

    Figure Lengend Snippet: Preparation of HDL associated LCAT. Size exclusion chromatography was perfo rmed at ambient temperature using a Superose-6 10/300 column on a Bio-Rad FPLC system (Hercules, CA). A 200 µl aliquot of plasma was injected per run, and eluted in PBS with 1 mM EDTA at a flow rate of 0.2 ml/min. Total cholesterol in each fraction was measured using the Cholesterol E Kit. LCAT activity was determined as following: A. 20 µl of each fraction of human plasma were incubated at 37ºC for 5 h; B. 2.5 µl of each fraction of WT mouse plasma were incubated at 37ºC for 2 h; C. 10 µl of each fraction of apoE-null mouse plasma were incubated at 37ºC for 4 h.

    Article Snippet: Preparation of HDL associated LCAT enzyme Fresh human or mouse blood was drawn into tubes containing potassium EDTA (Becton Dickinson, Franklin lakes, NJ), and plasma was separated immediately by low-speed centrifugation at 2500 rpm (1430g ) for 30 min at 4ºC.

    Techniques: Size-exclusion Chromatography, Fast Protein Liquid Chromatography, Injection, Flow Cytometry, Activity Assay, Incubation

    IFT27 directly interacts with nucleotide-empty ARL6 (A) LAP eluates from IFT27 LAP and LAP IFT88 were analyzed by immunoblotting for IFT-B subunits (IFT88, IFT57), IFT27 and ARL6. Twice as much of the LAP IFT88 eluate was loaded compared to the IFT27 LAP eluate. Note that the IFT27 antibody preferentially recognizes murine IFT27 over human IFT27, accounting for the lower signal intensity of IFT27 S-tag in the IFT27 LAP lane compared to that of murine IFT27 in the LAP IFT88 lane. (B) Co-transfections/co-immunoprecipitations were performed with all combinations of “GTP-locked” or “GDP-locked” variants of ARL6 and IFT27. IFT25 was co-transfected with IFT27 LAP to ensure IFT27 stability. (C) IFT25/IFT27 LAP -decorated beads were used to capture overexpressed Myc ARL6 out of HEK cell lysate in the presence or absence of EDTA. (D) The IFT25/IFT27-GST complex, GST, ARL6 and SAR1A were expressed in bacteria and purified to near-homogeneity before SDS-PAGE and Coomassie staining (left panel). IFT25/IFT27-GST was mixed with ARL6 or SAR1A in the presence of various nucleotides and complexes were recovered on Glutathione Sepharose beads before LDS elution, SDS-PAGE and Coomassie staining (middle panel). In a similar experiment, IFT25/IFT27-GST was mixed with ARL6 in the presence of GTPγS, EDTA or GDP/AlF 4 − (right panel). (E) ARL6, either alone or mixed with the IFT25/IFT27 complex and EDTA was resolved by size exclusion chromatography (Superdex 200). Size markers: 66.9 kDa (Thyroglobulin), 35.0 kDa (β-lactoglobulin) and 6.5 kDa (Aprotinin). .

    Journal: Developmental cell

    Article Title: The Intraflagellar Transport Protein IFT27 Promotes BBSome Exit from Cilia through the GTPase ARL6/BBS3

    doi: 10.1016/j.devcel.2014.09.004

    Figure Lengend Snippet: IFT27 directly interacts with nucleotide-empty ARL6 (A) LAP eluates from IFT27 LAP and LAP IFT88 were analyzed by immunoblotting for IFT-B subunits (IFT88, IFT57), IFT27 and ARL6. Twice as much of the LAP IFT88 eluate was loaded compared to the IFT27 LAP eluate. Note that the IFT27 antibody preferentially recognizes murine IFT27 over human IFT27, accounting for the lower signal intensity of IFT27 S-tag in the IFT27 LAP lane compared to that of murine IFT27 in the LAP IFT88 lane. (B) Co-transfections/co-immunoprecipitations were performed with all combinations of “GTP-locked” or “GDP-locked” variants of ARL6 and IFT27. IFT25 was co-transfected with IFT27 LAP to ensure IFT27 stability. (C) IFT25/IFT27 LAP -decorated beads were used to capture overexpressed Myc ARL6 out of HEK cell lysate in the presence or absence of EDTA. (D) The IFT25/IFT27-GST complex, GST, ARL6 and SAR1A were expressed in bacteria and purified to near-homogeneity before SDS-PAGE and Coomassie staining (left panel). IFT25/IFT27-GST was mixed with ARL6 or SAR1A in the presence of various nucleotides and complexes were recovered on Glutathione Sepharose beads before LDS elution, SDS-PAGE and Coomassie staining (middle panel). In a similar experiment, IFT25/IFT27-GST was mixed with ARL6 in the presence of GTPγS, EDTA or GDP/AlF 4 − (right panel). (E) ARL6, either alone or mixed with the IFT25/IFT27 complex and EDTA was resolved by size exclusion chromatography (Superdex 200). Size markers: 66.9 kDa (Thyroglobulin), 35.0 kDa (β-lactoglobulin) and 6.5 kDa (Aprotinin). .

    Article Snippet: 20 μM ARL6 was mixed on ice with 20 μM EDTA and 10 to 200 μM of IFT25/IFT27 variants in a total reaction volume of 300 μL and then transferred into a pre-heated quartz cuvette (Bio-Rad, #170-2504).

    Techniques: Transfection, Purification, SDS Page, Staining, Size-exclusion Chromatography

    IFT27 stabilizes the nucleotide-empty form of ARL6 (A) Time course of [ 3 for a summary of all conditions tested. (B–D) EDTA-induced ARL6 precipitation at 37°C was followed by light diffraction at 350 nm. (B) Rescue of ARL6 precipitation by stoichiometric concentrations of IFT25/ IFT27-GST. (C) Effect of different IFT25/IFT27 variants on the rescue of EDTA-induced ARL6 precipitation. Judging by endpoint absorbance values, IFT25/GST-IFT27 (N-terminal GST tag) was ~6 times more efficient than IFT25/IFT27-GST (C-terminal GST tag) in rescuing EDTA-induced Arl6 precipitation. Addition of IFT25 (even at 10-fold molar excess over ARL6) does not rescue precipitation of nucleotide empty ARL6. (D) Model for IFT27 stabilization of nucleotide-empty ARL6. .

    Journal: Developmental cell

    Article Title: The Intraflagellar Transport Protein IFT27 Promotes BBSome Exit from Cilia through the GTPase ARL6/BBS3

    doi: 10.1016/j.devcel.2014.09.004

    Figure Lengend Snippet: IFT27 stabilizes the nucleotide-empty form of ARL6 (A) Time course of [ 3 for a summary of all conditions tested. (B–D) EDTA-induced ARL6 precipitation at 37°C was followed by light diffraction at 350 nm. (B) Rescue of ARL6 precipitation by stoichiometric concentrations of IFT25/ IFT27-GST. (C) Effect of different IFT25/IFT27 variants on the rescue of EDTA-induced ARL6 precipitation. Judging by endpoint absorbance values, IFT25/GST-IFT27 (N-terminal GST tag) was ~6 times more efficient than IFT25/IFT27-GST (C-terminal GST tag) in rescuing EDTA-induced Arl6 precipitation. Addition of IFT25 (even at 10-fold molar excess over ARL6) does not rescue precipitation of nucleotide empty ARL6. (D) Model for IFT27 stabilization of nucleotide-empty ARL6. .

    Article Snippet: 20 μM ARL6 was mixed on ice with 20 μM EDTA and 10 to 200 μM of IFT25/IFT27 variants in a total reaction volume of 300 μL and then transferred into a pre-heated quartz cuvette (Bio-Rad, #170-2504).

    Techniques:

    Study 3: Comparison of PBMC isolation methods and SMN measures from various blood fractions. Due to the variability in SMN signal and protein concentrations seen with CPT tube PBMC isolation, other methods were explored using the four subjects from Study 2. A : PBMC yield was greatest in samples collected with EDTA tubes and subsequent Lymphoprep gradient separation, and showed no statistically significant changes with isolation delays of up to 24 h. B : Total soluble protein tended to increase with 24 h delays in samples collected by EDTA tube and isolated by CPT tubes, while there was no obvious change in protein concentrations in EDTA/Lymphoprep processing. C : SMN as measured by total protein tended to decrease with isolation delays with EDTA/CPT processing. SMN signals were similar with delays up to 24 h with EDTA/Lymphoprep processing. D : SMN by PBMC counts was variable for both EDTA/CPT and EDTA/Lymphoprep processing methods. However, the EDTA/Lymphoprep values were generally overlapping and did not appear to decrease from the no-delay timepoint (t = 0). In Figure 3 body of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median.

    Journal: PLoS ONE

    Article Title: Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein

    doi: 10.1371/journal.pone.0050763

    Figure Lengend Snippet: Study 3: Comparison of PBMC isolation methods and SMN measures from various blood fractions. Due to the variability in SMN signal and protein concentrations seen with CPT tube PBMC isolation, other methods were explored using the four subjects from Study 2. A : PBMC yield was greatest in samples collected with EDTA tubes and subsequent Lymphoprep gradient separation, and showed no statistically significant changes with isolation delays of up to 24 h. B : Total soluble protein tended to increase with 24 h delays in samples collected by EDTA tube and isolated by CPT tubes, while there was no obvious change in protein concentrations in EDTA/Lymphoprep processing. C : SMN as measured by total protein tended to decrease with isolation delays with EDTA/CPT processing. SMN signals were similar with delays up to 24 h with EDTA/Lymphoprep processing. D : SMN by PBMC counts was variable for both EDTA/CPT and EDTA/Lymphoprep processing methods. However, the EDTA/Lymphoprep values were generally overlapping and did not appear to decrease from the no-delay timepoint (t = 0). In Figure 3 body of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median.

    Article Snippet: Whole blood samples at the Jasper Clinic were collected in BD CPT tubes (#362761, Studies 1–3), EDTA tubes (#22-040-67, Fisher, Pittsburgh, PA in Studies 3–5, and SMAF-001) or ACD tubes (#363083, BD, Franklin Lakes, NJ in Study 5).

    Techniques: Isolation, Cycling Probe Technology

    Characterization of barcoded 601 (BC-601) DNA a , BC-601 DNA prepared for all 115 nucleosome library members as described in Methods (Barcoded 601 (BC-601) DNA preparation). Ligation products are 192 bp in size and were visualized by polyacrylamide gel electrophoresis (5% acrylamide, 0.5× TBE, 200 V, 40 min) and staining with SYBR Safe DNA gel stain. A faint band corresponding to unligated 601 DNA (601) is slightly visible in certain cases. b .

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Characterization of barcoded 601 (BC-601) DNA a , BC-601 DNA prepared for all 115 nucleosome library members as described in Methods (Barcoded 601 (BC-601) DNA preparation). Ligation products are 192 bp in size and were visualized by polyacrylamide gel electrophoresis (5% acrylamide, 0.5× TBE, 200 V, 40 min) and staining with SYBR Safe DNA gel stain. A faint band corresponding to unligated 601 DNA (601) is slightly visible in certain cases. b .

    Article Snippet: Quenched assay samples were directly run on a 5% TBE gel in 0.5× TBE buffer for 40 min at 200 V. Nucleosomes were visualized by staining with SYBR Gold Nucleic Acid Gel Stain (ThermoFisher Scientific).

    Techniques: Ligation, Polyacrylamide Gel Electrophoresis, Staining

    In vitro characterization of neuronal cell sheet derived from human induced pluripotent stem cells (hiPSCs). (A) A schematic representation of neuronal sheet formation from hiPSCs and transplantation. Embryoid bodies were developed from undifferentiated hiPSCs in a floating condition for 4 d. Then the cells were cultured in fibronectin-coated dishes for 4 d during which retinoic acid (RA), noggin (NOG), and cyclopamine (CyP; 3 factors) were introduced twice (on days 5 and 7). In some experiments, sonic hedgehog (SHH) were introduced instead of adding CyP, together with RA and NOG in order to compare the effects of CyP with those of SHH. Thereafter, the cells cultured with RA, NOG, and CyP were harvested on day 8. An aliquot of the cells was cultured in 24-well culture plates for 12 to 16 d and then harvested using trypsin/ethylenediaminetetraacetic acid (EDTA) solution for transplantation. The remaining cells were cultured on temperature-responsive gelation polymer-coated plate (temp. resp. polymer) for 12 to 16 d depending on the neuronal maturation, where they extended axon-like processes, leading to the formation of neuronal cell sheet. The neuronal cell sheet was recovered by lowering the temperature of culture plate below 22°C. Schedule for brain injury, transplantation, and motor function test was reported previously. 10 , 20 , 28 , 29 The cells on day 8 were used as neural stem/progenitor cells for comparison. (B) Stereomicroscopic view of the neuronal cell sheet cultured in a 48-well (diameter 12 mm) culture plate. Right lower part of the sheet detached from the bottom of the plate. (C) Stereomicroscopic view of the sheet in a 10-cm culture dish (diameter 90 mm). (D, E) Hematoxylin and eosin staining of the neuronal cell sheet (D, lower; E, higher magnification). Flow cytometric analysis of the cells in the sheet treated with trypsin/EDTA revealed that more than 75% of the cells expressed human NCAM (data not shown). (F, G) The neuronal cell sheet at day 19 was stained with anti-human nuclei (F) and anti-β-tubulin antibodies (G). (H) Real-time polymerase chain reaction analyses of the Figure 1. (continued) . neuronal cell sheet. For comparison, neural cells on day 8 (neural cells were cultured in the same manner except that SHH was added instead of CyP and were harvested on day 8 before making cell sheet) were included. The cell sheet expressed Foxp2, Fezf2, Igfbp4, CTIP2, and synaptophysin mRNA predominantly. CRIM1 mRNA was expressed throughout the culture period. Thus, the cell sheet expressed mRNAs of cortical motoneuron–associated proteins. A white horizontal bar represents 200 μm in panel D, 100 μm in panels E and F, and 50 μm in panel G. UiPSCs, undifferentiated-induced pluripotent stem cells; CRIM1, cysteine-rich motor neuron 1 protein precursor; MEF, mouse embryonic fibroblasts; floating indicates floating condition; Foxp2, forkhead box p2; Fezf2, forebrain embryonic zinc finger family zinc finger 2; Igfbp4, insulin-like growth factor-binding protein 4; CTIP2, COUP-TF-interacting protein 2; mRNA, messenger RNA.

    Journal: Cell Transplantation

    Article Title: Neuronal Cell Sheets of Cortical Motor Neuron Phenotype Derived from Human iPSCs

    doi: 10.1177/0963689717720280

    Figure Lengend Snippet: In vitro characterization of neuronal cell sheet derived from human induced pluripotent stem cells (hiPSCs). (A) A schematic representation of neuronal sheet formation from hiPSCs and transplantation. Embryoid bodies were developed from undifferentiated hiPSCs in a floating condition for 4 d. Then the cells were cultured in fibronectin-coated dishes for 4 d during which retinoic acid (RA), noggin (NOG), and cyclopamine (CyP; 3 factors) were introduced twice (on days 5 and 7). In some experiments, sonic hedgehog (SHH) were introduced instead of adding CyP, together with RA and NOG in order to compare the effects of CyP with those of SHH. Thereafter, the cells cultured with RA, NOG, and CyP were harvested on day 8. An aliquot of the cells was cultured in 24-well culture plates for 12 to 16 d and then harvested using trypsin/ethylenediaminetetraacetic acid (EDTA) solution for transplantation. The remaining cells were cultured on temperature-responsive gelation polymer-coated plate (temp. resp. polymer) for 12 to 16 d depending on the neuronal maturation, where they extended axon-like processes, leading to the formation of neuronal cell sheet. The neuronal cell sheet was recovered by lowering the temperature of culture plate below 22°C. Schedule for brain injury, transplantation, and motor function test was reported previously. 10 , 20 , 28 , 29 The cells on day 8 were used as neural stem/progenitor cells for comparison. (B) Stereomicroscopic view of the neuronal cell sheet cultured in a 48-well (diameter 12 mm) culture plate. Right lower part of the sheet detached from the bottom of the plate. (C) Stereomicroscopic view of the sheet in a 10-cm culture dish (diameter 90 mm). (D, E) Hematoxylin and eosin staining of the neuronal cell sheet (D, lower; E, higher magnification). Flow cytometric analysis of the cells in the sheet treated with trypsin/EDTA revealed that more than 75% of the cells expressed human NCAM (data not shown). (F, G) The neuronal cell sheet at day 19 was stained with anti-human nuclei (F) and anti-β-tubulin antibodies (G). (H) Real-time polymerase chain reaction analyses of the Figure 1. (continued) . neuronal cell sheet. For comparison, neural cells on day 8 (neural cells were cultured in the same manner except that SHH was added instead of CyP and were harvested on day 8 before making cell sheet) were included. The cell sheet expressed Foxp2, Fezf2, Igfbp4, CTIP2, and synaptophysin mRNA predominantly. CRIM1 mRNA was expressed throughout the culture period. Thus, the cell sheet expressed mRNAs of cortical motoneuron–associated proteins. A white horizontal bar represents 200 μm in panel D, 100 μm in panels E and F, and 50 μm in panel G. UiPSCs, undifferentiated-induced pluripotent stem cells; CRIM1, cysteine-rich motor neuron 1 protein precursor; MEF, mouse embryonic fibroblasts; floating indicates floating condition; Foxp2, forkhead box p2; Fezf2, forebrain embryonic zinc finger family zinc finger 2; Igfbp4, insulin-like growth factor-binding protein 4; CTIP2, COUP-TF-interacting protein 2; mRNA, messenger RNA.

    Article Snippet: An aliquot of the cells was cultured in 24-well culture plates for 12–16 d, which were then harvested using trypsin/ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Inc.) solution for transplantation.

    Techniques: In Vitro, Derivative Assay, Transplantation Assay, Cell Culture, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Binding Assay

    Ecto-tagged β1 integrins bind soluble ligand, restore surface levels of endogenous α5 integrins, and display normal activation indices. a Flow cytometry histograms showing binding of soluble FN9-10 to parental and a subset of reconstituted KO fibroblasts, in native conditions (filled orange peak ), EDTA-inhibited conditions ( blue peak ), or Mn 2 + -treated conditions ( red peak ). Full data set in Supplementary Fig. 3a . b , c Quantification of surface levels of human β1 ( b ) and mouse α5 integrins ( c ) measured by flow cytometry on parental and reconstituted β1 integrin fl/fl and KO fibroblasts. d Activation index of surface α5β1 integrins on parental and reconstituted β1 integrin fl/fl and KO fibroblasts, calculated as (FN9-10 binding in native conditions—FN9-10 binding in EDTA-inhibited conditions)/surface levels of α5 integrins. All data b – d is shown as mean ± SEM from four independent experiments. Statistical analysis was performed using one-way repeated measures ANOVA with Dunnett post hoc test. Each column was compared to KO no-tag β1 and * p

    Journal: Nature Communications

    Article Title: Novel ecto-tagged integrins reveal their trafficking in live cells

    doi: 10.1038/s41467-017-00646-w

    Figure Lengend Snippet: Ecto-tagged β1 integrins bind soluble ligand, restore surface levels of endogenous α5 integrins, and display normal activation indices. a Flow cytometry histograms showing binding of soluble FN9-10 to parental and a subset of reconstituted KO fibroblasts, in native conditions (filled orange peak ), EDTA-inhibited conditions ( blue peak ), or Mn 2 + -treated conditions ( red peak ). Full data set in Supplementary Fig. 3a . b , c Quantification of surface levels of human β1 ( b ) and mouse α5 integrins ( c ) measured by flow cytometry on parental and reconstituted β1 integrin fl/fl and KO fibroblasts. d Activation index of surface α5β1 integrins on parental and reconstituted β1 integrin fl/fl and KO fibroblasts, calculated as (FN9-10 binding in native conditions—FN9-10 binding in EDTA-inhibited conditions)/surface levels of α5 integrins. All data b – d is shown as mean ± SEM from four independent experiments. Statistical analysis was performed using one-way repeated measures ANOVA with Dunnett post hoc test. Each column was compared to KO no-tag β1 and * p

    Article Snippet: Mammalian cells were suspended with trypsin/EDTA, incubated with FN9-10 in the presence or absence of 10 mM EDTA or 0.5 mM MnCl2 , fixed with 4% paraformaldehyde, incubated with APC-Strepatavidin and analyzed by flow cytometry on an LSRII instrument (BD Biosciences).

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Binding Assay

    Kinetic analysis of circulating lymphocytes following influenza virus infection. Three ferrets each were inoculated i.n. with 10 6 PFU of virus. Blood was collected on days 3 (A), 7 (B), and 14 to 16 (C) p.i. in EDTA Vacutainer tubes and analyzed with

    Journal: Journal of Virology

    Article Title: Pathogenesis and Transmission of Triple-Reassortant Swine H1N1 Influenza Viruses Isolated before the 2009 H1N1 Pandemic ▿

    doi: 10.1128/JVI.02231-10

    Figure Lengend Snippet: Kinetic analysis of circulating lymphocytes following influenza virus infection. Three ferrets each were inoculated i.n. with 10 6 PFU of virus. Blood was collected on days 3 (A), 7 (B), and 14 to 16 (C) p.i. in EDTA Vacutainer tubes and analyzed with

    Article Snippet: Complete blood counts were quantified from blood collected in EDTA Vacutainer tubes (BD, Franklin Lakes, NJ) using a Hemavet HV950FS instrument per the manufacturer's instructions (Drew Scientific, Inc., Oxford, CT).

    Techniques: Infection

    GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by EDTA, confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in PBS (pH 3.5).

    Journal: Nanoscale

    Article Title: Antioxidant Chemistry of Graphene-Based Materials and its Role in Oxidation Protection Technology

    doi: 10.1039/c4nr03275f

    Figure Lengend Snippet: GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by EDTA, confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in PBS (pH 3.5).

    Article Snippet: Hydrogen peroxide (H2 O2 ), phosphate buffered saline (PBS) (pH 7.4), and EDTA were purchased from Fisher Scientific Inc. (Pittsburgh, PA).

    Techniques: Generated, Electron Paramagnetic Resonance, Spectroscopy, Concentration Assay, Binding Assay

    Ethanol Precipitation Dissociates the Catalytic Subunit from the Regulatory Subunits of PP2A. The effect of ethanol precipitation on the molecular form of PP2A was determined following size exclusion chromatography in Tris–EDTA buffer containing 10 mM β -mercaptoethanol and 100 mM NaCl on a Sephacryl 300HR column of the crude S100 fraction (3.0 mg protein) and S100 fraction following ethanol precipitation, extraction, and ammonium sulfate precipitation to form the EtOH-AS65 fraction (0.15 mg protein). PP2Ac in the fractions was determined by measuring the phosphatase activity toward a phosphothreonine peptide. Phosphatase activity is reported as nmol Pi produced during a 10 min incubation period per 225 μL of the column fractions. γ -Globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) were employed as molecular weight markers

    Journal: Neurochemical research

    Article Title: Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A

    doi: 10.1007/s11064-010-0310-4

    Figure Lengend Snippet: Ethanol Precipitation Dissociates the Catalytic Subunit from the Regulatory Subunits of PP2A. The effect of ethanol precipitation on the molecular form of PP2A was determined following size exclusion chromatography in Tris–EDTA buffer containing 10 mM β -mercaptoethanol and 100 mM NaCl on a Sephacryl 300HR column of the crude S100 fraction (3.0 mg protein) and S100 fraction following ethanol precipitation, extraction, and ammonium sulfate precipitation to form the EtOH-AS65 fraction (0.15 mg protein). PP2Ac in the fractions was determined by measuring the phosphatase activity toward a phosphothreonine peptide. Phosphatase activity is reported as nmol Pi produced during a 10 min incubation period per 225 μL of the column fractions. γ -Globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) were employed as molecular weight markers

    Article Snippet: For each S100 fraction prepared, one whole brain was partially thawed and homogenized in a glass-Teflon homogenizer in 15 mL of Tris–EDTA buffer (50 mM Tris, 1 mM EDTA, 1 mM benzamidine, pH 7.4) to which was added 50 μL of mammalian protease inhibitor cocktail (Sigma Chemical) per brain.

    Techniques: Ethanol Precipitation, Size-exclusion Chromatography, Activity Assay, Produced, Incubation, Molecular Weight

    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.

    Journal: Nucleic Acids Research

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    doi: 10.1093/nar/gkq271

    Figure Lengend Snippet: GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.

    Article Snippet: After an incubation at 37°C for 30 min, the reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science), and were further incubated at 37°C for 15 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 1.3 V/cm at 4°C for 16 h. The products were visualized by SYBR Gold (Invitrogen) staining.

    Techniques: Incubation, Labeling, Agarose Gel Electrophoresis, Imaging, Recombinase Polymerase Amplification, Staining, Standard Deviation