dna loading dye  (New England Biolabs)


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    New England Biolabs dna loading dye
    Targeted delivery of <t>DNA/RNA</t> payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask <t>dye</t> (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P
    Dna Loading Dye, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna loading dye/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna loading dye - by Bioz Stars, 2022-07
    94/100 stars

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    1) Product Images from "Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy"

    Article Title: Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

    Journal: Theranostics

    doi: 10.7150/thno.68667

    Targeted delivery of DNA/RNA payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask dye (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P
    Figure Legend Snippet: Targeted delivery of DNA/RNA payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask dye (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P

    Techniques Used: Flow Cytometry, Allele-specific Oligonucleotide, Fluorescence, Incubation, Staining, Expressing, Quantitative RT-PCR, CCK-8 Assay, One-tailed Test

    Antibody conjugated RBCEVs show increased accumulation in target cells. RBCEVs were conjugated with biotinylated monoclonal antibodies or nanobodies via a biotinylated linker peptide and streptavidin . (A) Single EV flow cytometric analysis of monoclonal antibody conjugation on RBCEVs using the streptavidin-mediated conjugation method. (B) Copy number of isotype control monoclonal antibody (mAb) and nanobodies (EGFR VHH) per RBCEV quantified using ELISA by comparison to a standard curve of antibodies/nanobodies (n = 6-9 replicates). (C) Flow cytometry analysis of CFSE in EGFR-positive CA1a cells or EGFR-negative MOLM13 cells treated with CFSE-labeled RBCEVs coated with control or EGFR-targeting nanobodies. (D) Representative immunofluorescent images of EV uptake in the co-culture of 4T1 and 4T1-tdTomato-hEGFR cells incubated with different RBCEV treatments. RBCEVs were tracked using CFSE (green), tdTomato was shown in red while nuclei were co-stained with Hoechst (blue). Scale bar is 20 µm. (E) Percentage difference in RBCEV uptake between 4T1-tdTomato-hEGFR cells and parental 4T1 cells, expressed as a fraction of mean CFSE intensity for each RBCEV treatment. (F) Uptake of EpCAM-targeted or control CFSE-labelled RBCEVs by EpCAM-positive H358 cells or EpCAM-negative MOLM13 cells. (G) Representative immunofluorescent images of EpCAM-targeting and non-targeting RCBEV uptake by H358 cells as in (F) . RBCEV uptake was observed using CFSE (green). CellMask was used to label the cell membrane (red) and the nucleus was visualized using Hoechst (blue). Scale bar is 50 µm. (H) RT-qPCR quantification of miR-125b ASOs loaded per EV obtained via comparison of the total RNA extract from miR-125b ASO-loaded EVs to a standard curve of miR-125b ASO (n = 3 biological replicates). (I) Quantification of miR-125b ASOs loaded per individual EV obtained via native PAGE analysis of miR-125b ASO-loaded RBCEVs electrophoresed alongside a serial dilution of unloaded miR-125b ASO (n = 6 biological replicates) . (J) Representative native PAGE analysis used to assess the loading efficiency of ASOs into EVs. Each EV lane denotes a separate biological replicate prepared using EVs from 3 blood donors (D1-D3). Graphs A, C, E and F represent data from 3 biological replicates prepared from RBCEVs from independent donors. For quantification of RNA per EV in H I , NTA was used to obtain the number of input EVs, thereby providing an estimate of ASO copy number per EV. The graphs present the mean ± SEM. Student's one-tailed t-test: ns - not significant, ***P
    Figure Legend Snippet: Antibody conjugated RBCEVs show increased accumulation in target cells. RBCEVs were conjugated with biotinylated monoclonal antibodies or nanobodies via a biotinylated linker peptide and streptavidin . (A) Single EV flow cytometric analysis of monoclonal antibody conjugation on RBCEVs using the streptavidin-mediated conjugation method. (B) Copy number of isotype control monoclonal antibody (mAb) and nanobodies (EGFR VHH) per RBCEV quantified using ELISA by comparison to a standard curve of antibodies/nanobodies (n = 6-9 replicates). (C) Flow cytometry analysis of CFSE in EGFR-positive CA1a cells or EGFR-negative MOLM13 cells treated with CFSE-labeled RBCEVs coated with control or EGFR-targeting nanobodies. (D) Representative immunofluorescent images of EV uptake in the co-culture of 4T1 and 4T1-tdTomato-hEGFR cells incubated with different RBCEV treatments. RBCEVs were tracked using CFSE (green), tdTomato was shown in red while nuclei were co-stained with Hoechst (blue). Scale bar is 20 µm. (E) Percentage difference in RBCEV uptake between 4T1-tdTomato-hEGFR cells and parental 4T1 cells, expressed as a fraction of mean CFSE intensity for each RBCEV treatment. (F) Uptake of EpCAM-targeted or control CFSE-labelled RBCEVs by EpCAM-positive H358 cells or EpCAM-negative MOLM13 cells. (G) Representative immunofluorescent images of EpCAM-targeting and non-targeting RCBEV uptake by H358 cells as in (F) . RBCEV uptake was observed using CFSE (green). CellMask was used to label the cell membrane (red) and the nucleus was visualized using Hoechst (blue). Scale bar is 50 µm. (H) RT-qPCR quantification of miR-125b ASOs loaded per EV obtained via comparison of the total RNA extract from miR-125b ASO-loaded EVs to a standard curve of miR-125b ASO (n = 3 biological replicates). (I) Quantification of miR-125b ASOs loaded per individual EV obtained via native PAGE analysis of miR-125b ASO-loaded RBCEVs electrophoresed alongside a serial dilution of unloaded miR-125b ASO (n = 6 biological replicates) . (J) Representative native PAGE analysis used to assess the loading efficiency of ASOs into EVs. Each EV lane denotes a separate biological replicate prepared using EVs from 3 blood donors (D1-D3). Graphs A, C, E and F represent data from 3 biological replicates prepared from RBCEVs from independent donors. For quantification of RNA per EV in H I , NTA was used to obtain the number of input EVs, thereby providing an estimate of ASO copy number per EV. The graphs present the mean ± SEM. Student's one-tailed t-test: ns - not significant, ***P

    Techniques Used: Conjugation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Labeling, Co-Culture Assay, Incubation, Staining, Quantitative RT-PCR, Allele-specific Oligonucleotide, Clear Native PAGE, Serial Dilution, One-tailed Test

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    New England Biolabs dna loading dye
    Targeted delivery of <t>DNA/RNA</t> payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask <t>dye</t> (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P
    Dna Loading Dye, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna loading dye/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna loading dye - by Bioz Stars, 2022-07
    94/100 stars
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    Targeted delivery of DNA/RNA payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask dye (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P

    Journal: Theranostics

    Article Title: Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

    doi: 10.7150/thno.68667

    Figure Lengend Snippet: Targeted delivery of DNA/RNA payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask dye (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P

    Article Snippet: The resulting homogenate was supplemented with 6 × DNA loading dye (New England Biolabs, USA), loaded onto 12% Native PAGE gel along with a serial dilution of input RNA and electrophoresed in TBE buffer for 1 hour.

    Techniques: Flow Cytometry, Allele-specific Oligonucleotide, Fluorescence, Incubation, Staining, Expressing, Quantitative RT-PCR, CCK-8 Assay, One-tailed Test

    Antibody conjugated RBCEVs show increased accumulation in target cells. RBCEVs were conjugated with biotinylated monoclonal antibodies or nanobodies via a biotinylated linker peptide and streptavidin . (A) Single EV flow cytometric analysis of monoclonal antibody conjugation on RBCEVs using the streptavidin-mediated conjugation method. (B) Copy number of isotype control monoclonal antibody (mAb) and nanobodies (EGFR VHH) per RBCEV quantified using ELISA by comparison to a standard curve of antibodies/nanobodies (n = 6-9 replicates). (C) Flow cytometry analysis of CFSE in EGFR-positive CA1a cells or EGFR-negative MOLM13 cells treated with CFSE-labeled RBCEVs coated with control or EGFR-targeting nanobodies. (D) Representative immunofluorescent images of EV uptake in the co-culture of 4T1 and 4T1-tdTomato-hEGFR cells incubated with different RBCEV treatments. RBCEVs were tracked using CFSE (green), tdTomato was shown in red while nuclei were co-stained with Hoechst (blue). Scale bar is 20 µm. (E) Percentage difference in RBCEV uptake between 4T1-tdTomato-hEGFR cells and parental 4T1 cells, expressed as a fraction of mean CFSE intensity for each RBCEV treatment. (F) Uptake of EpCAM-targeted or control CFSE-labelled RBCEVs by EpCAM-positive H358 cells or EpCAM-negative MOLM13 cells. (G) Representative immunofluorescent images of EpCAM-targeting and non-targeting RCBEV uptake by H358 cells as in (F) . RBCEV uptake was observed using CFSE (green). CellMask was used to label the cell membrane (red) and the nucleus was visualized using Hoechst (blue). Scale bar is 50 µm. (H) RT-qPCR quantification of miR-125b ASOs loaded per EV obtained via comparison of the total RNA extract from miR-125b ASO-loaded EVs to a standard curve of miR-125b ASO (n = 3 biological replicates). (I) Quantification of miR-125b ASOs loaded per individual EV obtained via native PAGE analysis of miR-125b ASO-loaded RBCEVs electrophoresed alongside a serial dilution of unloaded miR-125b ASO (n = 6 biological replicates) . (J) Representative native PAGE analysis used to assess the loading efficiency of ASOs into EVs. Each EV lane denotes a separate biological replicate prepared using EVs from 3 blood donors (D1-D3). Graphs A, C, E and F represent data from 3 biological replicates prepared from RBCEVs from independent donors. For quantification of RNA per EV in H I , NTA was used to obtain the number of input EVs, thereby providing an estimate of ASO copy number per EV. The graphs present the mean ± SEM. Student's one-tailed t-test: ns - not significant, ***P

    Journal: Theranostics

    Article Title: Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

    doi: 10.7150/thno.68667

    Figure Lengend Snippet: Antibody conjugated RBCEVs show increased accumulation in target cells. RBCEVs were conjugated with biotinylated monoclonal antibodies or nanobodies via a biotinylated linker peptide and streptavidin . (A) Single EV flow cytometric analysis of monoclonal antibody conjugation on RBCEVs using the streptavidin-mediated conjugation method. (B) Copy number of isotype control monoclonal antibody (mAb) and nanobodies (EGFR VHH) per RBCEV quantified using ELISA by comparison to a standard curve of antibodies/nanobodies (n = 6-9 replicates). (C) Flow cytometry analysis of CFSE in EGFR-positive CA1a cells or EGFR-negative MOLM13 cells treated with CFSE-labeled RBCEVs coated with control or EGFR-targeting nanobodies. (D) Representative immunofluorescent images of EV uptake in the co-culture of 4T1 and 4T1-tdTomato-hEGFR cells incubated with different RBCEV treatments. RBCEVs were tracked using CFSE (green), tdTomato was shown in red while nuclei were co-stained with Hoechst (blue). Scale bar is 20 µm. (E) Percentage difference in RBCEV uptake between 4T1-tdTomato-hEGFR cells and parental 4T1 cells, expressed as a fraction of mean CFSE intensity for each RBCEV treatment. (F) Uptake of EpCAM-targeted or control CFSE-labelled RBCEVs by EpCAM-positive H358 cells or EpCAM-negative MOLM13 cells. (G) Representative immunofluorescent images of EpCAM-targeting and non-targeting RCBEV uptake by H358 cells as in (F) . RBCEV uptake was observed using CFSE (green). CellMask was used to label the cell membrane (red) and the nucleus was visualized using Hoechst (blue). Scale bar is 50 µm. (H) RT-qPCR quantification of miR-125b ASOs loaded per EV obtained via comparison of the total RNA extract from miR-125b ASO-loaded EVs to a standard curve of miR-125b ASO (n = 3 biological replicates). (I) Quantification of miR-125b ASOs loaded per individual EV obtained via native PAGE analysis of miR-125b ASO-loaded RBCEVs electrophoresed alongside a serial dilution of unloaded miR-125b ASO (n = 6 biological replicates) . (J) Representative native PAGE analysis used to assess the loading efficiency of ASOs into EVs. Each EV lane denotes a separate biological replicate prepared using EVs from 3 blood donors (D1-D3). Graphs A, C, E and F represent data from 3 biological replicates prepared from RBCEVs from independent donors. For quantification of RNA per EV in H I , NTA was used to obtain the number of input EVs, thereby providing an estimate of ASO copy number per EV. The graphs present the mean ± SEM. Student's one-tailed t-test: ns - not significant, ***P

    Article Snippet: The resulting homogenate was supplemented with 6 × DNA loading dye (New England Biolabs, USA), loaded onto 12% Native PAGE gel along with a serial dilution of input RNA and electrophoresed in TBE buffer for 1 hour.

    Techniques: Conjugation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Labeling, Co-Culture Assay, Incubation, Staining, Quantitative RT-PCR, Allele-specific Oligonucleotide, Clear Native PAGE, Serial Dilution, One-tailed Test