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  • 99
    Thermo Fisher trypsinedta
    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 <t>trypsin-EDTA</t> 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 <t>cytometry</t> ( 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
    Trypsinedta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 83 article reviews
    Price from $9.99 to $1999.99
    trypsinedta - by Bioz Stars, 2020-08
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    99
    Millipore trypsinedta
    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 <t>trypsin-EDTA</t> 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 <t>cytometry</t> ( 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
    Trypsinedta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trypsinedta/product/Millipore
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
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    94
    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: 94/100, based on 3526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trypsin edta solution/product/Millipore
    Average 94 stars, based on 3526 article reviews
    Price from $9.99 to $1999.99
    trypsin edta solution - by Bioz Stars, 2020-08
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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
    https://www.bioz.com/result/trypsin edta/product/Millipore
    Average 99 stars, based on 10138 article reviews
    Price from $9.99 to $1999.99
    trypsin edta - by Bioz Stars, 2020-08
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    94
    Millipore trypsin edta solution 10x
    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 Solution 10x, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    trypsin edta solution 10x - by Bioz Stars, 2020-08
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    Image Search Results


    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

    Comparison of original and modified protocols for the ex vivo cultivation of human oral mucosal epithelial cells (OMECs). Left panel: In the original protocol, oral epithelium was isolated from oral mucosa with dispase II. Suspended OMECs were then dissociated with trypsin-EDTA, re-suspended and cultivated on de-epithelialized amniotic membrane (AM) with 3T3 fibroblasts as a feeder layer, in SHEM containing fetal bovine serum (FBS). Right panel: In the modified protocol, oral mucosal tissues were treated with collagenase A. The resulting cell aggregates were grown on AM without a 3T3 feeder layer. In addition, FBS was replaced with PLTMax. Two days after seeding, the culture medium was changed to serum-free medium (EpiLife) to prevent the overgrowth of submucosal fibroblasts.

    Journal: Scientific Reports

    Article Title: Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation

    doi: 10.1038/srep36266

    Figure Lengend Snippet: Comparison of original and modified protocols for the ex vivo cultivation of human oral mucosal epithelial cells (OMECs). Left panel: In the original protocol, oral epithelium was isolated from oral mucosa with dispase II. Suspended OMECs were then dissociated with trypsin-EDTA, re-suspended and cultivated on de-epithelialized amniotic membrane (AM) with 3T3 fibroblasts as a feeder layer, in SHEM containing fetal bovine serum (FBS). Right panel: In the modified protocol, oral mucosal tissues were treated with collagenase A. The resulting cell aggregates were grown on AM without a 3T3 feeder layer. In addition, FBS was replaced with PLTMax. Two days after seeding, the culture medium was changed to serum-free medium (EpiLife) to prevent the overgrowth of submucosal fibroblasts.

    Article Snippet: Materials DMEM:F12 (1:1), Opti-MEM, EpiLife medium, , trypsin-EDTA, phosphate-buffered saline (PBS), gentamicin, amphotericin B, Lipofectamine 2000 and Alexa-Fluor-conjugated secondary IgG were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Modification, Ex Vivo, Isolation

    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

    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