dt40  (ATCC)


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    ATCC dt40
    Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to <t>DT40</t> cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.
    Dt40, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro"

    Article Title: Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro

    Journal: Viruses

    doi: 10.3390/v15010130

    Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.
    Figure Legend Snippet: Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.

    Techniques Used: Negative Control, Incubation, Sequencing

    Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log 10 TCID 50 /mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.
    Figure Legend Snippet: Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log 10 TCID 50 /mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.

    Techniques Used: Mutagenesis, Negative Control

    dt40  (ATCC)


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    ATCC dt40
    Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to <t>DT40</t> cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.
    Dt40, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro"

    Article Title: Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro

    Journal: Viruses

    doi: 10.3390/v15010130

    Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.
    Figure Legend Snippet: Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.

    Techniques Used: Negative Control, Incubation, Sequencing

    Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log 10 TCID 50 /mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.
    Figure Legend Snippet: Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log 10 TCID 50 /mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.

    Techniques Used: Mutagenesis, Negative Control

    chicken b cell lymphoma cell line  (ATCC)


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    ATCC chicken b cell lymphoma cell line
    Chicken B Cell Lymphoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    blij 2111  (ATCC)


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    ATCC blij 2111
    Blij 2111, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dt40  (ATCC)


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    ATCC dt40
    The vv IBDV field strain UK661 was serially diluted ten-fold from 1:100 to 1:100,000, and the diluted stocks were frozen at −80 °C. Each diluted stock was subsequently thawed and subject to titration by TCID 50 in <t>DT40</t> cells, and by EID 50 in embyronated chicken eggs, and a linear regression analysis was performed (A). Hyperimmune serum from birds inoculated with the IBDV vaccine strain 2512 (genogroup A1) was obtained from Charles River. The serum was heat inactivated, serially diluted two-fold, and mixed with 100 TCID 50 of UK661. The mixture was added to DT40 cells in quadruplicate, and after 3 days, wells were fixed and stained with an antibody against the IBDV VP3 protein and a secondary antibody conjugated to a fluorophore. The wells were either scored positive or negative for the presence of IBDV antigen by immunofluorescence microscopy, and the percentage of positive cells in each well was quantified by flow cytometry to calculate the titer of the neutralising antibodies in the serum (B), compared to control serum from SPF birds that did not contain antibodies against IBDV (C). Each point represents the % of VP3+ DT40 cells in one well, the bar represents the mean, and error bars represent the standard deviation of the mean. The horizontal dashed line represents the limit of detection by TCID 50 (B and C). Plasmids pBG98A and pPBG98B were electroporated to DT40 cells, and cell cultures were fed with fresh DT40 cells every 72 hours. Cells were fixed and stained with an antibody against IBDV VP3, a secondary antibody conjugated to AlexaFluor 568, and the nuclei were stained with DAPI. The IBDV antigen was present in the cytoplasm of the DT40 cells (VP3 viral antigen in red, DAPI in blue) (D). At each passage, the supernatant of the cultures was harvested, and serially diluted 10-fold in additional DT40 cells, to determine the titer as described by Reed & Muench. Three biological repeats were titrated and the mean titer plotted for each passage. Error bars represent the standard deviation of the mean (E).
    Dt40, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Evaluating the breadth of neutralizing antibody responses elicited by infectious bursal disease virus (IBDV) genogroup A1 strains using a novel chicken B-cell rescue system and neutralization assay"

    Article Title: Evaluating the breadth of neutralizing antibody responses elicited by infectious bursal disease virus (IBDV) genogroup A1 strains using a novel chicken B-cell rescue system and neutralization assay

    Journal: bioRxiv

    doi: 10.1101/2022.06.03.494759

    The vv IBDV field strain UK661 was serially diluted ten-fold from 1:100 to 1:100,000, and the diluted stocks were frozen at −80 °C. Each diluted stock was subsequently thawed and subject to titration by TCID 50 in DT40 cells, and by EID 50 in embyronated chicken eggs, and a linear regression analysis was performed (A). Hyperimmune serum from birds inoculated with the IBDV vaccine strain 2512 (genogroup A1) was obtained from Charles River. The serum was heat inactivated, serially diluted two-fold, and mixed with 100 TCID 50 of UK661. The mixture was added to DT40 cells in quadruplicate, and after 3 days, wells were fixed and stained with an antibody against the IBDV VP3 protein and a secondary antibody conjugated to a fluorophore. The wells were either scored positive or negative for the presence of IBDV antigen by immunofluorescence microscopy, and the percentage of positive cells in each well was quantified by flow cytometry to calculate the titer of the neutralising antibodies in the serum (B), compared to control serum from SPF birds that did not contain antibodies against IBDV (C). Each point represents the % of VP3+ DT40 cells in one well, the bar represents the mean, and error bars represent the standard deviation of the mean. The horizontal dashed line represents the limit of detection by TCID 50 (B and C). Plasmids pBG98A and pPBG98B were electroporated to DT40 cells, and cell cultures were fed with fresh DT40 cells every 72 hours. Cells were fixed and stained with an antibody against IBDV VP3, a secondary antibody conjugated to AlexaFluor 568, and the nuclei were stained with DAPI. The IBDV antigen was present in the cytoplasm of the DT40 cells (VP3 viral antigen in red, DAPI in blue) (D). At each passage, the supernatant of the cultures was harvested, and serially diluted 10-fold in additional DT40 cells, to determine the titer as described by Reed & Muench. Three biological repeats were titrated and the mean titer plotted for each passage. Error bars represent the standard deviation of the mean (E).
    Figure Legend Snippet: The vv IBDV field strain UK661 was serially diluted ten-fold from 1:100 to 1:100,000, and the diluted stocks were frozen at −80 °C. Each diluted stock was subsequently thawed and subject to titration by TCID 50 in DT40 cells, and by EID 50 in embyronated chicken eggs, and a linear regression analysis was performed (A). Hyperimmune serum from birds inoculated with the IBDV vaccine strain 2512 (genogroup A1) was obtained from Charles River. The serum was heat inactivated, serially diluted two-fold, and mixed with 100 TCID 50 of UK661. The mixture was added to DT40 cells in quadruplicate, and after 3 days, wells were fixed and stained with an antibody against the IBDV VP3 protein and a secondary antibody conjugated to a fluorophore. The wells were either scored positive or negative for the presence of IBDV antigen by immunofluorescence microscopy, and the percentage of positive cells in each well was quantified by flow cytometry to calculate the titer of the neutralising antibodies in the serum (B), compared to control serum from SPF birds that did not contain antibodies against IBDV (C). Each point represents the % of VP3+ DT40 cells in one well, the bar represents the mean, and error bars represent the standard deviation of the mean. The horizontal dashed line represents the limit of detection by TCID 50 (B and C). Plasmids pBG98A and pPBG98B were electroporated to DT40 cells, and cell cultures were fed with fresh DT40 cells every 72 hours. Cells were fixed and stained with an antibody against IBDV VP3, a secondary antibody conjugated to AlexaFluor 568, and the nuclei were stained with DAPI. The IBDV antigen was present in the cytoplasm of the DT40 cells (VP3 viral antigen in red, DAPI in blue) (D). At each passage, the supernatant of the cultures was harvested, and serially diluted 10-fold in additional DT40 cells, to determine the titer as described by Reed & Muench. Three biological repeats were titrated and the mean titer plotted for each passage. Error bars represent the standard deviation of the mean (E).

    Techniques Used: Titration, Staining, Immunofluorescence, Microscopy, Flow Cytometry, Standard Deviation

    Reverse genetics plasmids encoding segment A of the lab-adapted strain PBG98 (pPBG98A) were designed where the 333 nucleotides that encode the 111 amino acids (residues 220 to 330) of the HVR were swapped for one of seven field strains (pPBG98A-HVR-VP2s of 7 IBDVs). The plasmids were co-electroporated with the reverse genetics plasmid encoding segment B (pPBG98B) into DT40 cells to rescue the viruses (A). The HVR amino acid sequences from the seven field strains F52-70, Del-E, SHG19, UK661, M04/09, ITA-04 and Vic-01/94 were aligned with PBG98. The accession numbers and genogroup numbers are given in parenthesis. Conserved residues are depicted as black dots and different residues are highlighted in red (B). The replication kinetics of the seven recombinant chimeric IBDVs was determined in triplicate by titration of infected cell supernatants at 12, 24 and 48 hpi in DT40 cells, expressed as log 10 TCID 50 /mL, and the mean plotted (error bars represent standard deviation of the mean) (C).
    Figure Legend Snippet: Reverse genetics plasmids encoding segment A of the lab-adapted strain PBG98 (pPBG98A) were designed where the 333 nucleotides that encode the 111 amino acids (residues 220 to 330) of the HVR were swapped for one of seven field strains (pPBG98A-HVR-VP2s of 7 IBDVs). The plasmids were co-electroporated with the reverse genetics plasmid encoding segment B (pPBG98B) into DT40 cells to rescue the viruses (A). The HVR amino acid sequences from the seven field strains F52-70, Del-E, SHG19, UK661, M04/09, ITA-04 and Vic-01/94 were aligned with PBG98. The accession numbers and genogroup numbers are given in parenthesis. Conserved residues are depicted as black dots and different residues are highlighted in red (B). The replication kinetics of the seven recombinant chimeric IBDVs was determined in triplicate by titration of infected cell supernatants at 12, 24 and 48 hpi in DT40 cells, expressed as log 10 TCID 50 /mL, and the mean plotted (error bars represent standard deviation of the mean) (C).

    Techniques Used: Plasmid Preparation, Recombinant, Titration, Infection, Standard Deviation

    The nucleotide sequences of the HVRs encoded by the seven plasmids, and present in the seven rescued and DT40-passaged viruses, were compared to the sequences in GenBank (Accession numbers provided) for strains F52-70, Del-E, SHG19, UK661, M04/09, ITA-04 and Vic-01/94. For each strain, the sequence in GenBank is displayed and conserved residues in the plasmid and the rescued virus are depicted as black dots, and different residues were listed. The four hydrophilic loops (P-BC, P-DE, P-FG, and P-HI), important for antigenicity, are boxed. Mutations previously reported to be involved in the adaptation of IBDV to adherent cell culture (positions 253, 279, 284 and 330) are highlighted in blue, and other common variable positions are shaded in orange.
    Figure Legend Snippet: The nucleotide sequences of the HVRs encoded by the seven plasmids, and present in the seven rescued and DT40-passaged viruses, were compared to the sequences in GenBank (Accession numbers provided) for strains F52-70, Del-E, SHG19, UK661, M04/09, ITA-04 and Vic-01/94. For each strain, the sequence in GenBank is displayed and conserved residues in the plasmid and the rescued virus are depicted as black dots, and different residues were listed. The four hydrophilic loops (P-BC, P-DE, P-FG, and P-HI), important for antigenicity, are boxed. Mutations previously reported to be involved in the adaptation of IBDV to adherent cell culture (positions 253, 279, 284 and 330) are highlighted in blue, and other common variable positions are shaded in orange.

    Techniques Used: Sequencing, Plasmid Preparation, Cell Culture

    The predicted structure of the VP2 of IBDV strain PBG98 was modelled using AlphaFold, and the side view and axial view were displayed. The HVR was depicted as solid grey. The predicted structures of PBG98, F52-70, Del-E, SHG19, UK661, M04/09, ITA-04, and Vic-01/94 were modelled using AlphaFold, based on the sequence that were in GenBank. The structures were compared to the PBG98 HVR structure and amino acid differences highlighted in orange (A). The predicted structures of the HVRs of the DT40-passaged viruses F52-70, Del-E, SHG19, UK661, M04/09, ITA-04, and Vic-01/94 were modelled using AlphaFold, and residues that were different from the GenBank sequences were highlighted in purple (B).
    Figure Legend Snippet: The predicted structure of the VP2 of IBDV strain PBG98 was modelled using AlphaFold, and the side view and axial view were displayed. The HVR was depicted as solid grey. The predicted structures of PBG98, F52-70, Del-E, SHG19, UK661, M04/09, ITA-04, and Vic-01/94 were modelled using AlphaFold, based on the sequence that were in GenBank. The structures were compared to the PBG98 HVR structure and amino acid differences highlighted in orange (A). The predicted structures of the HVRs of the DT40-passaged viruses F52-70, Del-E, SHG19, UK661, M04/09, ITA-04, and Vic-01/94 were modelled using AlphaFold, and residues that were different from the GenBank sequences were highlighted in purple (B).

    Techniques Used: Sequencing

    Viral neutralization assays were performed in DT40 cells, and the highest dilution of serum where there were no IBDV VP3 antigen positive cells was considered as the VN titer, which was expressed as log 2 and plotted on a graph for birds inoculated with F52-70, closed circles (A) or 228E, open circles (B). The VN titer of the serum from three birds inoculated with F5270 and three inoculated with 228E was determined against chimeric-viruses containing the HVR from F52-70, Del-E, SHG19, UK661, M04/09, ITA-04, and Vic-01/94 and the backbone from PBG98 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
    Figure Legend Snippet: Viral neutralization assays were performed in DT40 cells, and the highest dilution of serum where there were no IBDV VP3 antigen positive cells was considered as the VN titer, which was expressed as log 2 and plotted on a graph for birds inoculated with F52-70, closed circles (A) or 228E, open circles (B). The VN titer of the serum from three birds inoculated with F5270 and three inoculated with 228E was determined against chimeric-viruses containing the HVR from F52-70, Del-E, SHG19, UK661, M04/09, ITA-04, and Vic-01/94 and the backbone from PBG98 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

    Techniques Used: Neutralization

    chicken b lymphocyte cell line dt40  (ATCC)


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    ATCC chicken b lymphocyte cell line dt40
    Chicken B Lymphocyte Cell Line Dt40, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dt40 cells  (ATCC)


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    ATCC dt40 cells
    Dt40 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    crl  (ATCC)


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    ATCC crl
    Crl, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    chicken dt40 cells  (ATCC)


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    ATCC chicken dt40 cells
    Proteomic profiling of synchronous mitotic entry (A) Workflow of time-resolved mitotic chromatin proteomics. <t>DT40</t> CDK1 as cells arrested in G 2 by 1NM-PP1 were released into mitosis by washout of the drug. Crosslinked cells were processed by ChEP and by LC-MS/MS ( <xref ref-type=Kustatscher et al., 2014b ). (B) Synchronous mitotic entry of DT40 CDK1 as cells. Cells were fixed with 4% formaldehyde and stained with Hoechst at the indicated times after 1NM-PP1 washout. Images are projections of z stacks. Scale bar, 10 μm. " width="250" height="auto" />
    Chicken Dt40 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mapping the invisible chromatin transactions of prophase chromosome remodeling"

    Article Title: Mapping the invisible chromatin transactions of prophase chromosome remodeling

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2021.12.039

    Proteomic profiling of synchronous mitotic entry (A) Workflow of time-resolved mitotic chromatin proteomics. DT40 CDK1 as cells arrested in G 2 by 1NM-PP1 were released into mitosis by washout of the drug. Crosslinked cells were processed by ChEP and by LC-MS/MS ( <xref ref-type=Kustatscher et al., 2014b ). (B) Synchronous mitotic entry of DT40 CDK1 as cells. Cells were fixed with 4% formaldehyde and stained with Hoechst at the indicated times after 1NM-PP1 washout. Images are projections of z stacks. Scale bar, 10 μm. " title="... entry (A) Workflow of time-resolved mitotic chromatin proteomics. DT40 CDK1 as cells arrested in G 2 by ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Proteomic profiling of synchronous mitotic entry (A) Workflow of time-resolved mitotic chromatin proteomics. DT40 CDK1 as cells arrested in G 2 by 1NM-PP1 were released into mitosis by washout of the drug. Crosslinked cells were processed by ChEP and by LC-MS/MS ( Kustatscher et al., 2014b ). (B) Synchronous mitotic entry of DT40 CDK1 as cells. Cells were fixed with 4% formaldehyde and stained with Hoechst at the indicated times after 1NM-PP1 washout. Images are projections of z stacks. Scale bar, 10 μm.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Staining

    Independent confirmation of proteomics results (A) Chromosome association with lamin B1 in early mitosis. Still images from live-cell imaging of a DT40 cell expressing lamin B1 halo. DNA was stained with SiR-DNA. A single z section is shown. Bar: 5 μm. (B–I) Stepwise removal or assembly of chromosomal proteins in early mitosis. Changes reflect differential reduction or accumulation of inner nuclear membrane, nucleolar, and kinetochore proteins in ChEP chromatin. Shown are (B) lamin B1, (D) RPF2 (a LSU component), and NOP58 (an SSU component), (F) ROD and NDC80, and (H) lamin B1 and lamin B receptor (LBR). A recombinant LBR protein with Clover tag was detected by anti-GFP antibody. Histones H3 and H4 are loading controls. (C), (E), (G), and (I) are quantification of proteins shown in (B), (D), (F), and (H), respectively.
    Figure Legend Snippet: Independent confirmation of proteomics results (A) Chromosome association with lamin B1 in early mitosis. Still images from live-cell imaging of a DT40 cell expressing lamin B1 halo. DNA was stained with SiR-DNA. A single z section is shown. Bar: 5 μm. (B–I) Stepwise removal or assembly of chromosomal proteins in early mitosis. Changes reflect differential reduction or accumulation of inner nuclear membrane, nucleolar, and kinetochore proteins in ChEP chromatin. Shown are (B) lamin B1, (D) RPF2 (a LSU component), and NOP58 (an SSU component), (F) ROD and NDC80, and (H) lamin B1 and lamin B receptor (LBR). A recombinant LBR protein with Clover tag was detected by anti-GFP antibody. Histones H3 and H4 are loading controls. (C), (E), (G), and (I) are quantification of proteins shown in (B), (D), (F), and (H), respectively.

    Techniques Used: Live Cell Imaging, Expressing, Staining, Recombinant


    Figure Legend Snippet:

    Techniques Used: Recombinant, dsDNA Assay, Transfection, Mass Spectrometry, Western Blot, Microscopy, Expressing, Sequencing, Plasmid Preparation, Knock-In, Construct, Software

    dt40 chicken lymphoma  (ATCC)


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    ATCC dt40 chicken lymphoma
    Dt40 Chicken Lymphoma, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nies 2111 plasmid2  (ATCC)


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    ATCC nies 2111 plasmid2
    Genomic region of Nostoc sp. PCC 7120 plasmid pCC7120alpha and Nostoc sp. <t>NIES-2111</t> <t>plasmid2</t> encoding homologs of the main protein involved in the plasmid conjugative apparatus [relaxase- alr7199 , virB4 - alr7206 (T4CP) and virD4 - alr7213 (T4SS)]. Previous studies characterized the mobility of plasmids , MOB v , and MPF c . See for annotation of the relaxase, T4CP, and T4SS proteins in the 185 genomes involved in the present work.
    Nies 2111 Plasmid2, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mining of Cyanobacterial Genomes Indicates Natural Product Biosynthetic Gene Clusters Located in Conjugative Plasmids"

    Article Title: Mining of Cyanobacterial Genomes Indicates Natural Product Biosynthetic Gene Clusters Located in Conjugative Plasmids

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.684565

    Genomic region of Nostoc sp. PCC 7120 plasmid pCC7120alpha and Nostoc sp. NIES-2111 plasmid2 encoding homologs of the main protein involved in the plasmid conjugative apparatus [relaxase- alr7199 , virB4 - alr7206 (T4CP) and virD4 - alr7213 (T4SS)]. Previous studies characterized the mobility of plasmids , MOB v , and MPF c . See for annotation of the relaxase, T4CP, and T4SS proteins in the 185 genomes involved in the present work.
    Figure Legend Snippet: Genomic region of Nostoc sp. PCC 7120 plasmid pCC7120alpha and Nostoc sp. NIES-2111 plasmid2 encoding homologs of the main protein involved in the plasmid conjugative apparatus [relaxase- alr7199 , virB4 - alr7206 (T4CP) and virD4 - alr7213 (T4SS)]. Previous studies characterized the mobility of plasmids , MOB v , and MPF c . See for annotation of the relaxase, T4CP, and T4SS proteins in the 185 genomes involved in the present work.

    Techniques Used: Plasmid Preparation

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    ATCC nies 2111 plasmid2
    Genomic region of Nostoc sp. PCC 7120 plasmid pCC7120alpha and Nostoc sp. <t>NIES-2111</t> <t>plasmid2</t> encoding homologs of the main protein involved in the plasmid conjugative apparatus [relaxase- alr7199 , virB4 - alr7206 (T4CP) and virD4 - alr7213 (T4SS)]. Previous studies characterized the mobility of plasmids , MOB v , and MPF c . See for annotation of the relaxase, T4CP, and T4SS proteins in the 185 genomes involved in the present work.
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    Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.

    Journal: Viruses

    Article Title: Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro

    doi: 10.3390/v15010130

    Figure Lengend Snippet: Generation of IBDV escape mutants. 100 TCID 50 of IBDV strain F52/70 was mixed with 1:5000 of either ( a ) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with ( b ) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID 50 assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.

    Article Snippet: The chicken B-cell lymphoma cell-line, DT40 (ATCC, catalog number CRL-2111), was maintained in RPMI media supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Gillingham, UK), 1% l-glutamine (Sigma-Aldrich), 10% tryptose phosphate broth (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), and 50 mM beta-mercaptoethanol (Gibco) (complete DT40 media).

    Techniques: Negative Control, Incubation, Sequencing

    Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log 10 TCID 50 /mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.

    Journal: Viruses

    Article Title: Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro

    doi: 10.3390/v15010130

    Figure Lengend Snippet: Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log 10 TCID 50 /mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.

    Article Snippet: The chicken B-cell lymphoma cell-line, DT40 (ATCC, catalog number CRL-2111), was maintained in RPMI media supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Gillingham, UK), 1% l-glutamine (Sigma-Aldrich), 10% tryptose phosphate broth (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), and 50 mM beta-mercaptoethanol (Gibco) (complete DT40 media).

    Techniques: Mutagenesis, Negative Control

    Proteomic profiling of synchronous mitotic entry (A) Workflow of time-resolved mitotic chromatin proteomics. DT40 CDK1 as cells arrested in G 2 by 1NM-PP1 were released into mitosis by washout of the drug. Crosslinked cells were processed by ChEP and by LC-MS/MS ( <xref ref-type=Kustatscher et al., 2014b ). (B) Synchronous mitotic entry of DT40 CDK1 as cells. Cells were fixed with 4% formaldehyde and stained with Hoechst at the indicated times after 1NM-PP1 washout. Images are projections of z stacks. Scale bar, 10 μm. " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: Mapping the invisible chromatin transactions of prophase chromosome remodeling

    doi: 10.1016/j.molcel.2021.12.039

    Figure Lengend Snippet: Proteomic profiling of synchronous mitotic entry (A) Workflow of time-resolved mitotic chromatin proteomics. DT40 CDK1 as cells arrested in G 2 by 1NM-PP1 were released into mitosis by washout of the drug. Crosslinked cells were processed by ChEP and by LC-MS/MS ( Kustatscher et al., 2014b ). (B) Synchronous mitotic entry of DT40 CDK1 as cells. Cells were fixed with 4% formaldehyde and stained with Hoechst at the indicated times after 1NM-PP1 washout. Images are projections of z stacks. Scale bar, 10 μm.

    Article Snippet: Chicken DT40 cells , ATCC , CRL-2111.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Staining

    Independent confirmation of proteomics results (A) Chromosome association with lamin B1 in early mitosis. Still images from live-cell imaging of a DT40 cell expressing lamin B1 halo. DNA was stained with SiR-DNA. A single z section is shown. Bar: 5 μm. (B–I) Stepwise removal or assembly of chromosomal proteins in early mitosis. Changes reflect differential reduction or accumulation of inner nuclear membrane, nucleolar, and kinetochore proteins in ChEP chromatin. Shown are (B) lamin B1, (D) RPF2 (a LSU component), and NOP58 (an SSU component), (F) ROD and NDC80, and (H) lamin B1 and lamin B receptor (LBR). A recombinant LBR protein with Clover tag was detected by anti-GFP antibody. Histones H3 and H4 are loading controls. (C), (E), (G), and (I) are quantification of proteins shown in (B), (D), (F), and (H), respectively.

    Journal: Molecular Cell

    Article Title: Mapping the invisible chromatin transactions of prophase chromosome remodeling

    doi: 10.1016/j.molcel.2021.12.039

    Figure Lengend Snippet: Independent confirmation of proteomics results (A) Chromosome association with lamin B1 in early mitosis. Still images from live-cell imaging of a DT40 cell expressing lamin B1 halo. DNA was stained with SiR-DNA. A single z section is shown. Bar: 5 μm. (B–I) Stepwise removal or assembly of chromosomal proteins in early mitosis. Changes reflect differential reduction or accumulation of inner nuclear membrane, nucleolar, and kinetochore proteins in ChEP chromatin. Shown are (B) lamin B1, (D) RPF2 (a LSU component), and NOP58 (an SSU component), (F) ROD and NDC80, and (H) lamin B1 and lamin B receptor (LBR). A recombinant LBR protein with Clover tag was detected by anti-GFP antibody. Histones H3 and H4 are loading controls. (C), (E), (G), and (I) are quantification of proteins shown in (B), (D), (F), and (H), respectively.

    Article Snippet: Chicken DT40 cells , ATCC , CRL-2111.

    Techniques: Live Cell Imaging, Expressing, Staining, Recombinant

    Journal: Molecular Cell

    Article Title: Mapping the invisible chromatin transactions of prophase chromosome remodeling

    doi: 10.1016/j.molcel.2021.12.039

    Figure Lengend Snippet:

    Article Snippet: Chicken DT40 cells , ATCC , CRL-2111.

    Techniques: Recombinant, dsDNA Assay, Transfection, Mass Spectrometry, Western Blot, Microscopy, Expressing, Sequencing, Plasmid Preparation, Knock-In, Construct, Software

    Genomic region of Nostoc sp. PCC 7120 plasmid pCC7120alpha and Nostoc sp. NIES-2111 plasmid2 encoding homologs of the main protein involved in the plasmid conjugative apparatus [relaxase- alr7199 , virB4 - alr7206 (T4CP) and virD4 - alr7213 (T4SS)]. Previous studies characterized the mobility of plasmids , MOB v , and MPF c . See for annotation of the relaxase, T4CP, and T4SS proteins in the 185 genomes involved in the present work.

    Journal: Frontiers in Microbiology

    Article Title: Mining of Cyanobacterial Genomes Indicates Natural Product Biosynthetic Gene Clusters Located in Conjugative Plasmids

    doi: 10.3389/fmicb.2021.684565

    Figure Lengend Snippet: Genomic region of Nostoc sp. PCC 7120 plasmid pCC7120alpha and Nostoc sp. NIES-2111 plasmid2 encoding homologs of the main protein involved in the plasmid conjugative apparatus [relaxase- alr7199 , virB4 - alr7206 (T4CP) and virD4 - alr7213 (T4SS)]. Previous studies characterized the mobility of plasmids , MOB v , and MPF c . See for annotation of the relaxase, T4CP, and T4SS proteins in the 185 genomes involved in the present work.

    Article Snippet: Other known natural product BGCs were located in smaller plasmids: i.e., aeruginosin in Cylindrospermum sp. NIES-4074 plasmid1 (340 kb); anabaenopeptin in Gloeothece citriformis PCC 7424 plasmid pP742401 (328 kb) and Gloeothece verrucose PCC 7822 plasmid Cy782201 (879 kb), cryptophycin in Nostoc sp. ATCC 53789 plasmid pNSP_c (219 kb), geosmin in Nostoc sp. NIES-2111 plasmid2 (320 kb), and hassallidin in Aulosira laxa NIES-50 plasmid1 (292 kb) and Tolypothrix tenuis PCC 7101 plasmid1 (292 kb) ( ).

    Techniques: Plasmid Preparation