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  • 86
    Becton Dickinson facsaria cell sorter
    Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, <t>FACSAria.</t> Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.
    Facsaria Cell Sorter, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson facsaria ii cell sorter
    Bone marrow B cell flow cytometry results from a healthy donor. First, B cells were gated by using specific cell surface markers that were CD138 + and CD38 + by determining forward and side light scattering characteristics on the <t>FACSAria</t> II Cell Sorter (Becton Dickinson, San Jose, CA, USA). Then sorted B cells using with cell sorting by the cell surface markers CD56 + , CD19 + according to the FACSAria II Cell Sorter.
    Facsaria Ii Cell Sorter, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson facsaria iii cell sorter
    HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD <t>FACSAria™</t> <t>III</t> cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p
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    99
    Becton Dickinson facs
    HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD <t>FACSAria™</t> <t>III</t> cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p
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    Image Search Results


    Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, FACSAria. Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, FACSAria. Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Generated, Transfection, RNA Binding Assay, Synthesized, Plasmid Preparation, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Expressing, Negative Control, Quantitative RT-PCR

    Induction of cell death using an inverted switch. ( a ) Schematic illustration of the experiment. Together with the INPUT plasmids (500 ng), we transfected two plasmids: one expressing anti-apoptotic gene, Bcl-xL (10 ng) and ON-Kt-B (or dKt, 100 ng), which outputs apoptotic gene, Bim-EL instead of EGFP. ( b ) Induction of annexin V-positive cells. For transfection, 20 ng of cognate and 480 ng of noncognate INPUT plasmids were mixed. The left panel shows histograms of Pacific Blue-labelled anti-annexin V using FACSAria. A black line indicates a threshold used in this analysis. The right panel shows a ratio of annexin V-positive cells to the transfected cells. Bars and error bars represent the mean and s.d., respectively, of two replicates.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Induction of cell death using an inverted switch. ( a ) Schematic illustration of the experiment. Together with the INPUT plasmids (500 ng), we transfected two plasmids: one expressing anti-apoptotic gene, Bcl-xL (10 ng) and ON-Kt-B (or dKt, 100 ng), which outputs apoptotic gene, Bim-EL instead of EGFP. ( b ) Induction of annexin V-positive cells. For transfection, 20 ng of cognate and 480 ng of noncognate INPUT plasmids were mixed. The left panel shows histograms of Pacific Blue-labelled anti-annexin V using FACSAria. A black line indicates a threshold used in this analysis. The right panel shows a ratio of annexin V-positive cells to the transfected cells. Bars and error bars represent the mean and s.d., respectively, of two replicates.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Transfection, Expressing

    Correlation of the sensitivity and reactivity between the inverted ON switches and parental OFF switches. ( a ) Behaviour of the switches as a function of the amount of input. The x a xis shows the ratio of the INPUT plasmids expressing cognate RNA-binding proteins to the OUTPUT plasmids for the indicated switches (100 ng). The total plasmid content was adjusted up to 600 ng using noncognate plasmids. Twenty-four hours after transfection, individual cells were analysed using FACSAria. Geometrical mean values of the ratio between EGFP and DsRed-Express signals in a cell were shown. ON-Fr15 and OFF-Fr15 are switches responsive to Bacillus ribosomal protein S15 instead of L7Ae. Data points and error bars represent the mean and s.d., respectively, of three replicates. See Supplementary Fig. S4 (ON-Kt and OFF-Kt) and Supplementary Fig. S5 (ON-Fr15 and OFF-Fr15) for plots produced from this analysis. ( b , c ) Western blotting analysis of input proteins is shown. Total protein from the transfected cells shown in a was extracted 24 h after transfection and subjected to western blotting analysis. Cognate (L7Ae ( b ) and S15 ( c )) and noncognate (MS2CP ( b ) and L7KK ( c )) input proteins were detected using anti-myc antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also analysed as an internal control of the lysates. The right three lanes were fivefold serial dilutions of the lysate prepared in the same condition as the ratio of INPUT plasmid/OUTPUT plasmid equal to 5. Representative result from the three independent experiments was shown. ( d ) Behaviour of the switches as a function of the affinity between the input protein and the corresponding sensory motif in the switch. See Supplementary Fig. S8 for plots generated from this analysis.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Correlation of the sensitivity and reactivity between the inverted ON switches and parental OFF switches. ( a ) Behaviour of the switches as a function of the amount of input. The x a xis shows the ratio of the INPUT plasmids expressing cognate RNA-binding proteins to the OUTPUT plasmids for the indicated switches (100 ng). The total plasmid content was adjusted up to 600 ng using noncognate plasmids. Twenty-four hours after transfection, individual cells were analysed using FACSAria. Geometrical mean values of the ratio between EGFP and DsRed-Express signals in a cell were shown. ON-Fr15 and OFF-Fr15 are switches responsive to Bacillus ribosomal protein S15 instead of L7Ae. Data points and error bars represent the mean and s.d., respectively, of three replicates. See Supplementary Fig. S4 (ON-Kt and OFF-Kt) and Supplementary Fig. S5 (ON-Fr15 and OFF-Fr15) for plots produced from this analysis. ( b , c ) Western blotting analysis of input proteins is shown. Total protein from the transfected cells shown in a was extracted 24 h after transfection and subjected to western blotting analysis. Cognate (L7Ae ( b ) and S15 ( c )) and noncognate (MS2CP ( b ) and L7KK ( c )) input proteins were detected using anti-myc antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also analysed as an internal control of the lysates. The right three lanes were fivefold serial dilutions of the lysate prepared in the same condition as the ratio of INPUT plasmid/OUTPUT plasmid equal to 5. Representative result from the three independent experiments was shown. ( d ) Behaviour of the switches as a function of the affinity between the input protein and the corresponding sensory motif in the switch. See Supplementary Fig. S8 for plots generated from this analysis.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Expressing, RNA Binding Assay, Plasmid Preparation, Transfection, Produced, Western Blot, Generated

    Simultaneous and symmetrical control of two outputs in a cell by using ON and OFF switches. ( a ) The behaviour of ON and OFF switches responsive to a single input (L7Ae). Two reporter proteins, EGFP and enhanced cyan fluorescent protein (ECFP), were produced from ON and OFF switches, respectively. Two OUTPUT plasmids expressing either an ON or an OFF switch (100 ng each) were transfected into HeLa cells together with 20 ng of cognate and 480 ng of noncognate INPUT plasmids. The fluorescent signals were normalized by those of the switches containing Kt in the absence of L7Ae. ( b ) Control of two switches under two input proteins. Kt or dKt in ON switches were replaced with Fr15 or dFr15 (defective Fr15). Two OUTPUT plasmids were transfected together with L7Ae- (20 ng) and/or S15- (480 ng) expressing plasmids. In this experiment, a viral nucleocapside protein was used as a noncognate RNA-binding protein. The fluorescent signals were normalized by those detected in the absence of both L7Ae and S15. Transfected cells were analysed using FACSAria. Bars and error bars represent the mean and s.d., respectively, of three replicates.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Simultaneous and symmetrical control of two outputs in a cell by using ON and OFF switches. ( a ) The behaviour of ON and OFF switches responsive to a single input (L7Ae). Two reporter proteins, EGFP and enhanced cyan fluorescent protein (ECFP), were produced from ON and OFF switches, respectively. Two OUTPUT plasmids expressing either an ON or an OFF switch (100 ng each) were transfected into HeLa cells together with 20 ng of cognate and 480 ng of noncognate INPUT plasmids. The fluorescent signals were normalized by those of the switches containing Kt in the absence of L7Ae. ( b ) Control of two switches under two input proteins. Kt or dKt in ON switches were replaced with Fr15 or dFr15 (defective Fr15). Two OUTPUT plasmids were transfected together with L7Ae- (20 ng) and/or S15- (480 ng) expressing plasmids. In this experiment, a viral nucleocapside protein was used as a noncognate RNA-binding protein. The fluorescent signals were normalized by those detected in the absence of both L7Ae and S15. Transfected cells were analysed using FACSAria. Bars and error bars represent the mean and s.d., respectively, of three replicates.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Produced, Expressing, Transfection, RNA Binding Assay

    Bone marrow B cell flow cytometry results from a healthy donor. First, B cells were gated by using specific cell surface markers that were CD138 + and CD38 + by determining forward and side light scattering characteristics on the FACSAria II Cell Sorter (Becton Dickinson, San Jose, CA, USA). Then sorted B cells using with cell sorting by the cell surface markers CD56 + , CD19 + according to the FACSAria II Cell Sorter.

    Journal: Balkan Medical Journal

    Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis

    doi: 10.4274/balkanmedj.2018.0356

    Figure Lengend Snippet: Bone marrow B cell flow cytometry results from a healthy donor. First, B cells were gated by using specific cell surface markers that were CD138 + and CD38 + by determining forward and side light scattering characteristics on the FACSAria II Cell Sorter (Becton Dickinson, San Jose, CA, USA). Then sorted B cells using with cell sorting by the cell surface markers CD56 + , CD19 + according to the FACSAria II Cell Sorter.

    Article Snippet: Fluorescence-activated cell sorting Myeloma cells (CD38+, CD138+, CD19-, and CD56+) and healthy B cells (CD38+, CD138+, CD19+, and CD56-) were selected from bone marrow mononuclear cells using a gating strategy by simultaneously specifying cell surface markers, and by determining forward and side light scattering characteristics on the FACSAria II Cell Sorter (Becton Dickinson, San Jose, CA, USA) ( , ).

    Techniques: Flow Cytometry, Cytometry, FACS

    Flow cytometry results of malignant B cells from bone marrow of a patient with Multiple myeloma. First, myeloma cells were gated by using specific cell surface markers that were CD138 + and CD38 + by determining forward and side light scattering characteristics on the FACSAria II Cell Sorter (Becton Dickinson, San Jose, CA, USA). Then sorted malignant Multiple myeloma cells using with cell sorting by the cell surface markers CD56+, CD19- according to the FACSAria II Cell Sorter.

    Journal: Balkan Medical Journal

    Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis

    doi: 10.4274/balkanmedj.2018.0356

    Figure Lengend Snippet: Flow cytometry results of malignant B cells from bone marrow of a patient with Multiple myeloma. First, myeloma cells were gated by using specific cell surface markers that were CD138 + and CD38 + by determining forward and side light scattering characteristics on the FACSAria II Cell Sorter (Becton Dickinson, San Jose, CA, USA). Then sorted malignant Multiple myeloma cells using with cell sorting by the cell surface markers CD56+, CD19- according to the FACSAria II Cell Sorter.

    Article Snippet: Fluorescence-activated cell sorting Myeloma cells (CD38+, CD138+, CD19-, and CD56+) and healthy B cells (CD38+, CD138+, CD19+, and CD56-) were selected from bone marrow mononuclear cells using a gating strategy by simultaneously specifying cell surface markers, and by determining forward and side light scattering characteristics on the FACSAria II Cell Sorter (Becton Dickinson, San Jose, CA, USA) ( , ).

    Techniques: Flow Cytometry, Cytometry, FACS

    HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p

    Journal: Viruses

    Article Title: Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

    doi: 10.3390/v8100285

    Figure Lengend Snippet: HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p

    Article Snippet: After several passages, infected cells were sorted using a BD FACSAria™ III Cell Sorter (BD Biosciences, San Jose, CA, USA) according to GFP emission since Cas9 protein was fused to GFP.

    Techniques: Expressing, Fluorescence, FACS

    BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.

    Journal: Viruses

    Article Title: Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

    doi: 10.3390/v8100285

    Figure Lengend Snippet: BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.

    Article Snippet: After several passages, infected cells were sorted using a BD FACSAria™ III Cell Sorter (BD Biosciences, San Jose, CA, USA) according to GFP emission since Cas9 protein was fused to GFP.

    Techniques: Förster Resonance Energy Transfer, Expressing, Construct, Variant Assay, Fluorescence, Transfection