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  • 99
    Thermo Fisher optimem medium
    DNA binding ability of fluorescent compounds was evaluated in <t>OptiMEM</t> by mixing a constant mass of DNA (1 μg) with different concentrations of cationic lipids corresponding to different CRs. DNA full complexation was highlighted by the absence of DNA migration into the agarose gel, at a given CR depending on the formulations tested.
    Optimem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher opti mem
    Stability of CS–ASO nanocomplexes in the <t>transfection</t> medium. The stability of CS–ASO nanocomplexes with ( a ) ASOgreen, ( b ) ASOgreen_sense and ( c ) 5′Fam-ASOgreen at a P/N charge ratio of 90 with 85 mM NaCl is shown. Nanocomplexes were incubated in <t>Opti-MEM™</t> or Opti-MEM™ supplemented with HEPES (20 mM) and mannitol (270 mM) at 37 °C. Nanocomplexes in Opti-MEM™ supplemented with HEPES and mannitol appeared to be more stable than nanocomplexes in Opti-MEM™ alone.
    Opti Mem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 56618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher optimem
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 21007 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Lonza opti mem medium
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Opti Mem Medium, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore opti mem medium
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Opti Mem Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shanghai Genechem opti mem medium
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Opti Mem Medium, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher serum medium optimem
    Optimal delivery of mod-mRNAs and m-miRNAs enhances the reprogramming of human primary fibroblasts. a Transfection efficiency (top) and mean fluorescence intensity (bottom) of human primary neonatal fibroblasts (FN2) transfected with 500 ng of mod-mRNA encoding mWasabi, using indicated transfection buffers, as determined by flow cytometry 24 h post transfection. Error bars, mean ± s.d. for all panels ( n = 3). The results are reproducible using an independent primary neonatal fibroblast line, FN1 (Supplementary Fig. 3a ). b Schematic diagram of the optimized RNA-based reprogramming regimen with RNAs delivered using <t>Opti-MEM</t> adjusted to a pH of 8.2 as the transfection buffer for RNAiMAX. c Effect of mod-mRNA titration and the addition of m-miRNAs on the reprogramming of human primary neonatal fibroblasts (FN2). All reprogramming conditions were initiated at 500 cells per well of a 6-well format dish. Cells were transfected every 48 h with differing amounts of mod-mRNAs encoding mWasabi (transfection control) and either d2eGFP as a negative control or 6-factor reprogramming cocktails containing either M 3 O (5fM 3 O) or OCT4 (5fOCT4). Mod-mRNA transfections were performed alone or in combination with m-miRNAs or AllStars Negative control siRNA (neg) transfections, using the indicated transfection buffers. The number of resulting TRA-1-60-positive colonies on day 18 of the indicated regimens are plotted. Error bars, mean ± s.d. ( n = 3). The yield of TRA-1-60-positive colonies was compared between the regimens performed in the presence or absence of m-miRNAs. P values were calculated using the unpaired two-tailed Student’s t test. ** P
    Serum Medium Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    EuroClone opti mem medium
    Optimal delivery of mod-mRNAs and m-miRNAs enhances the reprogramming of human primary fibroblasts. a Transfection efficiency (top) and mean fluorescence intensity (bottom) of human primary neonatal fibroblasts (FN2) transfected with 500 ng of mod-mRNA encoding mWasabi, using indicated transfection buffers, as determined by flow cytometry 24 h post transfection. Error bars, mean ± s.d. for all panels ( n = 3). The results are reproducible using an independent primary neonatal fibroblast line, FN1 (Supplementary Fig. 3a ). b Schematic diagram of the optimized RNA-based reprogramming regimen with RNAs delivered using <t>Opti-MEM</t> adjusted to a pH of 8.2 as the transfection buffer for RNAiMAX. c Effect of mod-mRNA titration and the addition of m-miRNAs on the reprogramming of human primary neonatal fibroblasts (FN2). All reprogramming conditions were initiated at 500 cells per well of a 6-well format dish. Cells were transfected every 48 h with differing amounts of mod-mRNAs encoding mWasabi (transfection control) and either d2eGFP as a negative control or 6-factor reprogramming cocktails containing either M 3 O (5fM 3 O) or OCT4 (5fOCT4). Mod-mRNA transfections were performed alone or in combination with m-miRNAs or AllStars Negative control siRNA (neg) transfections, using the indicated transfection buffers. The number of resulting TRA-1-60-positive colonies on day 18 of the indicated regimens are plotted. Error bars, mean ± s.d. ( n = 3). The yield of TRA-1-60-positive colonies was compared between the regimens performed in the presence or absence of m-miRNAs. P values were calculated using the unpaired two-tailed Student’s t test. ** P
    Opti Mem Medium, supplied by EuroClone, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA binding ability of fluorescent compounds was evaluated in OptiMEM by mixing a constant mass of DNA (1 μg) with different concentrations of cationic lipids corresponding to different CRs. DNA full complexation was highlighted by the absence of DNA migration into the agarose gel, at a given CR depending on the formulations tested.

    Journal: International Journal of Molecular Sciences

    Article Title: Evaluation of New Fluorescent Lipophosphoramidates for Gene Transfer and Biodistribution Studies after Systemic Administration

    doi: 10.3390/ijms161125941

    Figure Lengend Snippet: DNA binding ability of fluorescent compounds was evaluated in OptiMEM by mixing a constant mass of DNA (1 μg) with different concentrations of cationic lipids corresponding to different CRs. DNA full complexation was highlighted by the absence of DNA migration into the agarose gel, at a given CR depending on the formulations tested.

    Article Snippet: Various lipoplexes characterized by several CRs (1 to 8) were prepared with a constant amount of DNA plasmid (0.25 µg) in OptiMEM medium (Gibco).

    Techniques: Binding Assay, Migration, Agarose Gel Electrophoresis

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates c-kit and stem cell factor (SCF) expression by BM-derived DCs (BMdDCs) . (A–C) Effect of GM-CSF and cell density on c-kit expression by BMdDCs. BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium in four different conditions, that is at either 2 × 10 5 or 1.2 × 10 5 cell/well, and either with or without (w/o) GM-CSF at 20 ng/ml, as indicated. Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. (A) Typical flow cytometric profiles, showing CD40 and MHCII expression by BMdDCs. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines represent isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Summary of c-kit expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (D,E) Effect of GM-CSF on SCF expression by BMdDCs. (D) Cell lysates were prepared from day 0 BMdDCs and BMdDCs cultured for 2 days in 24-well plates at 1.2 × 10 5 cell/well in complete Opti-MEM medium with or w/o GM-CSF at 20 ng/ml, as indicated. SCF protein expression was analyzed by ELISA, testing 25 µg of cell lysate in 100 µl/well. Data are expressed as picograms per milliliter. Individual results from three experiments and average values (bar) are shown. (E) Day 0 and day 2 BMdDCs cultured in 24-well plates at 1.2 × 10 5 cell/well with or w/o GM-CSF at 20 ng/ml were analyzed by Real-Time PCR in triplicates. SCF mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments, in (D) N = 3 experiments, in (E) mean ± SD of four experiments (* P ≤ 0.05; ** P ≤ 0.01).

    Journal: Frontiers in Immunology

    Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

    doi: 10.3389/fimmu.2017.00147

    Figure Lengend Snippet: Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates c-kit and stem cell factor (SCF) expression by BM-derived DCs (BMdDCs) . (A–C) Effect of GM-CSF and cell density on c-kit expression by BMdDCs. BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium in four different conditions, that is at either 2 × 10 5 or 1.2 × 10 5 cell/well, and either with or without (w/o) GM-CSF at 20 ng/ml, as indicated. Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. (A) Typical flow cytometric profiles, showing CD40 and MHCII expression by BMdDCs. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines represent isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Summary of c-kit expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (D,E) Effect of GM-CSF on SCF expression by BMdDCs. (D) Cell lysates were prepared from day 0 BMdDCs and BMdDCs cultured for 2 days in 24-well plates at 1.2 × 10 5 cell/well in complete Opti-MEM medium with or w/o GM-CSF at 20 ng/ml, as indicated. SCF protein expression was analyzed by ELISA, testing 25 µg of cell lysate in 100 µl/well. Data are expressed as picograms per milliliter. Individual results from three experiments and average values (bar) are shown. (E) Day 0 and day 2 BMdDCs cultured in 24-well plates at 1.2 × 10 5 cell/well with or w/o GM-CSF at 20 ng/ml were analyzed by Real-Time PCR in triplicates. SCF mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments, in (D) N = 3 experiments, in (E) mean ± SD of four experiments (* P ≤ 0.05; ** P ≤ 0.01).

    Article Snippet: After 2 days of culture in complete Opti-MEM medium, the majority of BMdDCs were MHC-IIhi CD40hi CD11bint , and expressed c-kit+ (Figures A–C, bottom panels).

    Techniques: Expressing, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Negative effect of stem cell factor (SCF) silencing on BM-derived DC (BMdDC) survival . BMdDCs were transfected with either SCF-siRNA or control-scrambled (scrl) siRNA, or else left untreated. Cells cultured in triplicates in 96-well plates at 2 × 10 5 /well in complete Opti-MEM medium with or w/o granulocyte-macrophage colony-stimulating factor (GM-CSF) at 20 ng/ml were analyzed after 2 days. (A,B) Percentages of living cells. Flow cytometry analysis was performed after staining with Annexin V FITC and incubation with PI. (A) Typical Annexin V and PI staining profiles. Numbers represent percentages of cells in the indicated quadrants. Living cells are in the lower left quadrant (Annexin V − PI − ). (B) Summary of results obtained by analyzing living cell percentages among SCF-siRNA and control-scrambled siRNA treated BMdDCs, gated as in (A) . Percentages of living cells from individual samples and average values (bar). (C) Numbers of living cells. BMdDC numbers were evaluated by the CyQuant assay. Results of SCF-siRNA and control-scrambled siRNA treated samples were normalized over corresponding untreated BMdDCs. Numbers of living cells from individual samples and average values (bar). In (A) representative data of N = 5 experiments, in (B,C) N = 5 experiments (* P ≤ 0.05).

    Journal: Frontiers in Immunology

    Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

    doi: 10.3389/fimmu.2017.00147

    Figure Lengend Snippet: Negative effect of stem cell factor (SCF) silencing on BM-derived DC (BMdDC) survival . BMdDCs were transfected with either SCF-siRNA or control-scrambled (scrl) siRNA, or else left untreated. Cells cultured in triplicates in 96-well plates at 2 × 10 5 /well in complete Opti-MEM medium with or w/o granulocyte-macrophage colony-stimulating factor (GM-CSF) at 20 ng/ml were analyzed after 2 days. (A,B) Percentages of living cells. Flow cytometry analysis was performed after staining with Annexin V FITC and incubation with PI. (A) Typical Annexin V and PI staining profiles. Numbers represent percentages of cells in the indicated quadrants. Living cells are in the lower left quadrant (Annexin V − PI − ). (B) Summary of results obtained by analyzing living cell percentages among SCF-siRNA and control-scrambled siRNA treated BMdDCs, gated as in (A) . Percentages of living cells from individual samples and average values (bar). (C) Numbers of living cells. BMdDC numbers were evaluated by the CyQuant assay. Results of SCF-siRNA and control-scrambled siRNA treated samples were normalized over corresponding untreated BMdDCs. Numbers of living cells from individual samples and average values (bar). In (A) representative data of N = 5 experiments, in (B,C) N = 5 experiments (* P ≤ 0.05).

    Article Snippet: After 2 days of culture in complete Opti-MEM medium, the majority of BMdDCs were MHC-IIhi CD40hi CD11bint , and expressed c-kit+ (Figures A–C, bottom panels).

    Techniques: Derivative Assay, Transfection, Cell Culture, Flow Cytometry, Cytometry, Staining, Incubation, CyQUANT Assay

    BM-derived DCs (BMdDCs) express a functional c-kit receptor . BMdDCs were obtained by purifying CD11c + cells from bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 1 week, as explained in Section “ Materials and Methods ” (day 0). BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium with GM-CSF at 20 ng/ml (day 2). (A–D) Analysis of c-kit membrane expression by flow cytometry. Day 0 and day 2 BMdDCs were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry (for gating strategy, see Figure S3 in Supplementary Material). (A) Typical flow cytometric profiles showing CD40, CD11b, and MHCII expression by BMdDCs. In the left panels, numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Representative contour plots showing c-kit and CD11b expression by BMdDCs. (D) Summary of c-kit expression results obtained from day 0 and day 2 MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (E) Analysis of c-kit mRNA expression by Real-Time PCR. Day 0 and day 2 BMdDC samples were analyzed by Real-Time PCR in triplicates. c-kit mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. (F,G) Western blot analysis of phospho-AKT expression by BMdDCs stimulated with stem cell factor (SCF). Day 2 BMdDCs obtained as above were stimulated with SCF at 100 ng/ml for 5 and 15 min or left untreated, as indicated. Western blot was performed with anti-phospho-AKT, anti-AKT and anti-β actin mAbs, and results analyzed by densitometry. (F) Representative Western blot results. (G) Densitometric analysis. Phospho-AKT levels were calculated relative to AKT in arbitrary units. In (A–C) representative data of 9–16 experiments, in (D) N = 16 experiments, in (F) representative data of three experiments, in (E,G) mean ± SD of three experiments (* P ≤ 0.05; *** P ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

    doi: 10.3389/fimmu.2017.00147

    Figure Lengend Snippet: BM-derived DCs (BMdDCs) express a functional c-kit receptor . BMdDCs were obtained by purifying CD11c + cells from bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 1 week, as explained in Section “ Materials and Methods ” (day 0). BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium with GM-CSF at 20 ng/ml (day 2). (A–D) Analysis of c-kit membrane expression by flow cytometry. Day 0 and day 2 BMdDCs were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry (for gating strategy, see Figure S3 in Supplementary Material). (A) Typical flow cytometric profiles showing CD40, CD11b, and MHCII expression by BMdDCs. In the left panels, numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Representative contour plots showing c-kit and CD11b expression by BMdDCs. (D) Summary of c-kit expression results obtained from day 0 and day 2 MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (E) Analysis of c-kit mRNA expression by Real-Time PCR. Day 0 and day 2 BMdDC samples were analyzed by Real-Time PCR in triplicates. c-kit mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. (F,G) Western blot analysis of phospho-AKT expression by BMdDCs stimulated with stem cell factor (SCF). Day 2 BMdDCs obtained as above were stimulated with SCF at 100 ng/ml for 5 and 15 min or left untreated, as indicated. Western blot was performed with anti-phospho-AKT, anti-AKT and anti-β actin mAbs, and results analyzed by densitometry. (F) Representative Western blot results. (G) Densitometric analysis. Phospho-AKT levels were calculated relative to AKT in arbitrary units. In (A–C) representative data of 9–16 experiments, in (D) N = 16 experiments, in (F) representative data of three experiments, in (E,G) mean ± SD of three experiments (* P ≤ 0.05; *** P ≤ 0.001).

    Article Snippet: After 2 days of culture in complete Opti-MEM medium, the majority of BMdDCs were MHC-IIhi CD40hi CD11bint , and expressed c-kit+ (Figures A–C, bottom panels).

    Techniques: Derivative Assay, Functional Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Western Blot

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not stem cell factor (SCF) modulates CXCR4 expression by BM-derived DCs (BMdDCs) . BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium either with or without (w/o) GM-CSF at 20 ng/ml. SCF was then added at 100 ng/ml and cells were further incubated for 16 h. Cells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry. (A) Typical CD40 and MHCII expression profiles. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing CXCR4 expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent CXCR4 staining profiles, dashed lines represent no Ab (FMO, Fluorescence Minus One). Numbers indicate CXCR4 median fluorescence intensity (MFI) values. (C) Summary of CXCR4 expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . CXCR4 MFI from individual samples and average values (bar). In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments (** P ≤ 0.01; *** P ≤ 0.01).

    Journal: Frontiers in Immunology

    Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

    doi: 10.3389/fimmu.2017.00147

    Figure Lengend Snippet: Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not stem cell factor (SCF) modulates CXCR4 expression by BM-derived DCs (BMdDCs) . BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium either with or without (w/o) GM-CSF at 20 ng/ml. SCF was then added at 100 ng/ml and cells were further incubated for 16 h. Cells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry. (A) Typical CD40 and MHCII expression profiles. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing CXCR4 expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent CXCR4 staining profiles, dashed lines represent no Ab (FMO, Fluorescence Minus One). Numbers indicate CXCR4 median fluorescence intensity (MFI) values. (C) Summary of CXCR4 expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . CXCR4 MFI from individual samples and average values (bar). In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments (** P ≤ 0.01; *** P ≤ 0.01).

    Article Snippet: After 2 days of culture in complete Opti-MEM medium, the majority of BMdDCs were MHC-IIhi CD40hi CD11bint , and expressed c-kit+ (Figures A–C, bottom panels).

    Techniques: Expressing, Derivative Assay, Cell Culture, Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence

    Knockdown of PABPC1 with siRNA decreases PSA luciferase activity and PSA protein levels. (A) C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL), siPABPC1 (pool of 3 oligos) (40 pmol/mL), pRL-CMV (0.03μg), and PSA6.1Luc (0.3μg) in OPTI-MEM. The media was changed the next day to 5% chS FBS RPMI for 24 hours followed by treatment with 0.1nM R1881for an additional 24 hours in 5% chS FBS RPMI. Luciferase assay was performed with pRL-CMV (Renilla) luciferase used as a normalizer. (B) C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (40 pmol/mL) and treated as in (A) using increasing doses of R1881 (0, 0.1, and 1nM) followed by Western blot analysis. Blots were probed with antibodies specific for PABPC1 and PSA. GAPDH was used as a loading control. (C) C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (20 pmol/mL, 40 pmol/mL and 80 pmol/mL), in OPTI-MEM. Media was changed the next day to 10% FBS RPMI for 72 hours followed by Western blot analysis. Blots were probed as in (B). C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (40 pmol/mL), (D) or individual PABPC1 siRNA oligos (E) , pRL-CMV (0.03μg), and PSA6.1Luc (0.3μg) in OPTI-MEM. The media was changed the next day to 10% FBS RPMI for an additional 48 hours followed by luciferase assay. Experiments were repeated three times. Significance was determined by Student’s t-test (*p

    Journal: PLoS ONE

    Article Title: Poly (A) Binding Protein Cytoplasmic 1 Is a Novel Co-Regulator of the Androgen Receptor

    doi: 10.1371/journal.pone.0128495

    Figure Lengend Snippet: Knockdown of PABPC1 with siRNA decreases PSA luciferase activity and PSA protein levels. (A) C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL), siPABPC1 (pool of 3 oligos) (40 pmol/mL), pRL-CMV (0.03μg), and PSA6.1Luc (0.3μg) in OPTI-MEM. The media was changed the next day to 5% chS FBS RPMI for 24 hours followed by treatment with 0.1nM R1881for an additional 24 hours in 5% chS FBS RPMI. Luciferase assay was performed with pRL-CMV (Renilla) luciferase used as a normalizer. (B) C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (40 pmol/mL) and treated as in (A) using increasing doses of R1881 (0, 0.1, and 1nM) followed by Western blot analysis. Blots were probed with antibodies specific for PABPC1 and PSA. GAPDH was used as a loading control. (C) C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (20 pmol/mL, 40 pmol/mL and 80 pmol/mL), in OPTI-MEM. Media was changed the next day to 10% FBS RPMI for 72 hours followed by Western blot analysis. Blots were probed as in (B). C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (40 pmol/mL), (D) or individual PABPC1 siRNA oligos (E) , pRL-CMV (0.03μg), and PSA6.1Luc (0.3μg) in OPTI-MEM. The media was changed the next day to 10% FBS RPMI for an additional 48 hours followed by luciferase assay. Experiments were repeated three times. Significance was determined by Student’s t-test (*p

    Article Snippet: Cells were transfected in OPTI-MEM media (Life Technologies, Grand Island, NY) using Lipofectamine 2000 (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions and harvested 72 hours after transfection in RIPA buffer for analysis by Western blot using antibodies specific for PABPC1 (F-2), PSA (C-19) and GAPDH (FL-335) (all purchased from Santa Cruz Biotechnology, Dallas, TX).

    Techniques: Luciferase, Activity Assay, Transfection, Western Blot

    The effect of CdCl 2 on viability on Saos-2 cells. Cells were treated with 2.5,5, or 10 μM CdCl 2 in serum free Opti-MEM medium for 0, 3, 24, or 48hr (A) or 10, 50, or 100 μM CdCl 2 in McCoy’s 5A medium supplemented with 10% FBS for 0, 3, 24, or 48 hr (B). Controls received culture medium only. Cell viability was determined using the MTT assay. Results are expressed as percent of viable cells. Each line represents the mean ± SEM of at least 3–6 independent experiments. * denotes significant from control p

    Journal: Food and Chemical Toxicology

    Article Title: Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells

    doi: 10.1016/j.fct.2011.10.031

    Figure Lengend Snippet: The effect of CdCl 2 on viability on Saos-2 cells. Cells were treated with 2.5,5, or 10 μM CdCl 2 in serum free Opti-MEM medium for 0, 3, 24, or 48hr (A) or 10, 50, or 100 μM CdCl 2 in McCoy’s 5A medium supplemented with 10% FBS for 0, 3, 24, or 48 hr (B). Controls received culture medium only. Cell viability was determined using the MTT assay. Results are expressed as percent of viable cells. Each line represents the mean ± SEM of at least 3–6 independent experiments. * denotes significant from control p

    Article Snippet: Saos-2 cells were cultured in McCoy’s 5A medium (ATCC, Manassas, VA) or Opti-MEM serum free medium (Invitrogen, Carlsbad, CA).

    Techniques: MTT Assay

    IGFBP7 inhibits the growth of AML cells. ( a ) Average expression of IGFBP7 measured using qRT-PCR, error bars show the S.D. of a duplicate. ( b ) Lentivirally transduced leukemic cell lines were analysed for the expression of IGFBP7 by immunoblotting of the lysates (upper panel) and the medium (middle panel). The expression of actin was used as a control for equal loading (lower panel). * indicates an aspecific background band. ( c ) Cell viability of Kasumi-1 cells transduced with the control (EF-1) or with the IGFBP7 overexpression vector were cultured under low-serum conditions (1%) for 72 h. Bars and error bars show the cell viability and S.D. of a triplicate. ( d ) AML cells were treated with various concentrations of rhIGFBP7 in Opti-Mem reduced serum medium with 1% FCS for 72 h. Bars represent the average and error bars represent the viability and S.D. of a triplicate. One-way ANOVA analysis was used with a post hoc Tukey test to calculate the P -value * P ≤0.05, ** P ≤0.01, *** P ≤0.001

    Journal: Cell Death & Disease

    Article Title: IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

    doi: 10.1038/cddis.2014.268

    Figure Lengend Snippet: IGFBP7 inhibits the growth of AML cells. ( a ) Average expression of IGFBP7 measured using qRT-PCR, error bars show the S.D. of a duplicate. ( b ) Lentivirally transduced leukemic cell lines were analysed for the expression of IGFBP7 by immunoblotting of the lysates (upper panel) and the medium (middle panel). The expression of actin was used as a control for equal loading (lower panel). * indicates an aspecific background band. ( c ) Cell viability of Kasumi-1 cells transduced with the control (EF-1) or with the IGFBP7 overexpression vector were cultured under low-serum conditions (1%) for 72 h. Bars and error bars show the cell viability and S.D. of a triplicate. ( d ) AML cells were treated with various concentrations of rhIGFBP7 in Opti-Mem reduced serum medium with 1% FCS for 72 h. Bars represent the average and error bars represent the viability and S.D. of a triplicate. One-way ANOVA analysis was used with a post hoc Tukey test to calculate the P -value * P ≤0.05, ** P ≤0.01, *** P ≤0.001

    Article Snippet: Cells cultured under low-serum conditions were either cultured in RPMI-1640 with 1% FCS or in Opti-Mem Reduced-Serum Medium (used in the experiments illustrated in , and ; Life Technologies, Bleiswijk, The Netherlands) with 1% FCS.

    Techniques: Expressing, Quantitative RT-PCR, Transduction, Over Expression, Plasmid Preparation, Cell Culture

    Stability of CS–ASO nanocomplexes in the transfection medium. The stability of CS–ASO nanocomplexes with ( a ) ASOgreen, ( b ) ASOgreen_sense and ( c ) 5′Fam-ASOgreen at a P/N charge ratio of 90 with 85 mM NaCl is shown. Nanocomplexes were incubated in Opti-MEM™ or Opti-MEM™ supplemented with HEPES (20 mM) and mannitol (270 mM) at 37 °C. Nanocomplexes in Opti-MEM™ supplemented with HEPES and mannitol appeared to be more stable than nanocomplexes in Opti-MEM™ alone.

    Journal: Biomolecules

    Article Title: Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

    doi: 10.3390/biom10040553

    Figure Lengend Snippet: Stability of CS–ASO nanocomplexes in the transfection medium. The stability of CS–ASO nanocomplexes with ( a ) ASOgreen, ( b ) ASOgreen_sense and ( c ) 5′Fam-ASOgreen at a P/N charge ratio of 90 with 85 mM NaCl is shown. Nanocomplexes were incubated in Opti-MEM™ or Opti-MEM™ supplemented with HEPES (20 mM) and mannitol (270 mM) at 37 °C. Nanocomplexes in Opti-MEM™ supplemented with HEPES and mannitol appeared to be more stable than nanocomplexes in Opti-MEM™ alone.

    Article Snippet: Therefore, transfection experiments were conducted with Opti-MEM™ without supplements, even though nanocomplexes were more stable in the medium supplemented with HEPES and mannitol.

    Techniques: Allele-specific Oligonucleotide, Transfection, Incubation

    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Infection, Staining, Positive Control, Activity Assay

    Chemical inhibition of CME inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T monolayers were pretreated with chlorpromazine diluted in OptiMEM® for 30 min at 37°C. Cells were then infected with rVSV (MOI = 3) for 8 hrs and then analyzed as described in Figure 1 . Under these conditions, a MOI of 3 yielded 50-60% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. The rVSV-VSV G reporter virus was included as positive control. Results are expressed as percent virus-infected cells relative to that of controls and represent 3 independent experiments; error bars are SEM.

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: Chemical inhibition of CME inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T monolayers were pretreated with chlorpromazine diluted in OptiMEM® for 30 min at 37°C. Cells were then infected with rVSV (MOI = 3) for 8 hrs and then analyzed as described in Figure 1 . Under these conditions, a MOI of 3 yielded 50-60% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. The rVSV-VSV G reporter virus was included as positive control. Results are expressed as percent virus-infected cells relative to that of controls and represent 3 independent experiments; error bars are SEM.

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Inhibition, Infection, Staining, Positive Control

    CavME endocytosis and macropinocytosis are not required for ABLV G-mediated viral entry. (A) Chemical inhibition of CavME. HEK293T cell monolayers were pretreated with filipin diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection. (B) Cholera toxin B (CTX-B) subunit uptake is inhibited by filipin. Following pretreatment with filipin as described in (A) , HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 488-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI, (4′,6-diamidino-2-phenylindole, dihydrochloride). (C) Chemical inhibition of macropinocytosis. HEK293T cell monolayers were pretreated with EIPA diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . rVSV encoding VSV G (MOI = 1) or EboGP (MOI = 15) were included as negative and positive controls, respectively, to assess EIPA activity. Drug was maintained for the entire course of infection. For (A) and (C) results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are SEM. Under these experimental conditions, the chosen MOIs yielded 60-70% virus-infected cells in untreated controls. EIPA, 5-(N-ethyl-N-isopropyl) amiloride.

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: CavME endocytosis and macropinocytosis are not required for ABLV G-mediated viral entry. (A) Chemical inhibition of CavME. HEK293T cell monolayers were pretreated with filipin diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection. (B) Cholera toxin B (CTX-B) subunit uptake is inhibited by filipin. Following pretreatment with filipin as described in (A) , HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 488-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI, (4′,6-diamidino-2-phenylindole, dihydrochloride). (C) Chemical inhibition of macropinocytosis. HEK293T cell monolayers were pretreated with EIPA diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . rVSV encoding VSV G (MOI = 1) or EboGP (MOI = 15) were included as negative and positive controls, respectively, to assess EIPA activity. Drug was maintained for the entire course of infection. For (A) and (C) results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are SEM. Under these experimental conditions, the chosen MOIs yielded 60-70% virus-infected cells in untreated controls. EIPA, 5-(N-ethyl-N-isopropyl) amiloride.

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Inhibition, Infection, Incubation, Labeling, Confocal Microscopy, Staining, Activity Assay

    Chemical inhibition of dynamin inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T cell monolayers were pretreated with dynasore diluted in OptiMEM® for 30 min at 37°C and were then infected with max-GFP encoding rVSV reporter viruses (MOI = 1). Cells were harvested 20 hrs post infection, fixed with 2% paraformaldehyde and then analyzed for GFP expression (indicative of productive infection). GFP positive cells were counted with a Nexcelom Vision automated cell counter with fluorescence detection and the percent of virus-infected cells was calculated by dividing the number of GFP positive cells by the total number of cells counted. Under these experimental conditions, a MOI of 1 yielded 60-70% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess dynasore activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: Chemical inhibition of dynamin inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T cell monolayers were pretreated with dynasore diluted in OptiMEM® for 30 min at 37°C and were then infected with max-GFP encoding rVSV reporter viruses (MOI = 1). Cells were harvested 20 hrs post infection, fixed with 2% paraformaldehyde and then analyzed for GFP expression (indicative of productive infection). GFP positive cells were counted with a Nexcelom Vision automated cell counter with fluorescence detection and the percent of virus-infected cells was calculated by dividing the number of GFP positive cells by the total number of cells counted. Under these experimental conditions, a MOI of 1 yielded 60-70% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess dynasore activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Inhibition, Infection, Expressing, Fluorescence, Staining, Positive Control, Activity Assay

    Optimal delivery of mod-mRNAs and m-miRNAs enhances the reprogramming of human primary fibroblasts. a Transfection efficiency (top) and mean fluorescence intensity (bottom) of human primary neonatal fibroblasts (FN2) transfected with 500 ng of mod-mRNA encoding mWasabi, using indicated transfection buffers, as determined by flow cytometry 24 h post transfection. Error bars, mean ± s.d. for all panels ( n = 3). The results are reproducible using an independent primary neonatal fibroblast line, FN1 (Supplementary Fig. 3a ). b Schematic diagram of the optimized RNA-based reprogramming regimen with RNAs delivered using Opti-MEM adjusted to a pH of 8.2 as the transfection buffer for RNAiMAX. c Effect of mod-mRNA titration and the addition of m-miRNAs on the reprogramming of human primary neonatal fibroblasts (FN2). All reprogramming conditions were initiated at 500 cells per well of a 6-well format dish. Cells were transfected every 48 h with differing amounts of mod-mRNAs encoding mWasabi (transfection control) and either d2eGFP as a negative control or 6-factor reprogramming cocktails containing either M 3 O (5fM 3 O) or OCT4 (5fOCT4). Mod-mRNA transfections were performed alone or in combination with m-miRNAs or AllStars Negative control siRNA (neg) transfections, using the indicated transfection buffers. The number of resulting TRA-1-60-positive colonies on day 18 of the indicated regimens are plotted. Error bars, mean ± s.d. ( n = 3). The yield of TRA-1-60-positive colonies was compared between the regimens performed in the presence or absence of m-miRNAs. P values were calculated using the unpaired two-tailed Student’s t test. ** P

    Journal: Nature Communications

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts

    doi: 10.1038/s41467-018-03190-3

    Figure Lengend Snippet: Optimal delivery of mod-mRNAs and m-miRNAs enhances the reprogramming of human primary fibroblasts. a Transfection efficiency (top) and mean fluorescence intensity (bottom) of human primary neonatal fibroblasts (FN2) transfected with 500 ng of mod-mRNA encoding mWasabi, using indicated transfection buffers, as determined by flow cytometry 24 h post transfection. Error bars, mean ± s.d. for all panels ( n = 3). The results are reproducible using an independent primary neonatal fibroblast line, FN1 (Supplementary Fig. 3a ). b Schematic diagram of the optimized RNA-based reprogramming regimen with RNAs delivered using Opti-MEM adjusted to a pH of 8.2 as the transfection buffer for RNAiMAX. c Effect of mod-mRNA titration and the addition of m-miRNAs on the reprogramming of human primary neonatal fibroblasts (FN2). All reprogramming conditions were initiated at 500 cells per well of a 6-well format dish. Cells were transfected every 48 h with differing amounts of mod-mRNAs encoding mWasabi (transfection control) and either d2eGFP as a negative control or 6-factor reprogramming cocktails containing either M 3 O (5fM 3 O) or OCT4 (5fOCT4). Mod-mRNA transfections were performed alone or in combination with m-miRNAs or AllStars Negative control siRNA (neg) transfections, using the indicated transfection buffers. The number of resulting TRA-1-60-positive colonies on day 18 of the indicated regimens are plotted. Error bars, mean ± s.d. ( n = 3). The yield of TRA-1-60-positive colonies was compared between the regimens performed in the presence or absence of m-miRNAs. P values were calculated using the unpaired two-tailed Student’s t test. ** P

    Article Snippet: RNA and RNAiMAX were first diluted in either pH-adjusted Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific) or 1× pH-adjusted PBS as indicated in the corresponding figures. pH-adjusted Opti-MEM or pH-adjusted PBS were used as transfection buffers for complex formation between RNAiMAX and RNA.

    Techniques: Transfection, Fluorescence, Flow Cytometry, Cytometry, Titration, Negative Control, Two Tailed Test

    Defining the minimal and the optimal number of RNA transfections for the RNA-based reprogramming approach. Summary plots and representative TRA-1-60-stained reprogramming wells show the yield of TRA-1-60-positive colonies at day 18 of reprogramming initiated at a plating density of 500 cells per well of a 6-well format dish with two independent human primary neonatal fibroblast lines, FN1 (top) and FN2 (bottom). The reprogramming was accomplished using the indicated numbers of mod-mRNA and m-miRNA transfections performed at either 48 or 72 h intervals using Opti-MEM-8.2 as the transfection buffer. The amount of m-miRNA used per transfection is 20 pmol, and the amount of mod-mRNAs is indicated. Error bars, mean ± s.d. ( n = 3). No statistical difference in the reprogramming efficiencies was observed between 6 and 7 transfections (NS, P > 0.05). P values were calculated using the unpaired two-tailed Student’s t test. Scale bars, 10 mm

    Journal: Nature Communications

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts

    doi: 10.1038/s41467-018-03190-3

    Figure Lengend Snippet: Defining the minimal and the optimal number of RNA transfections for the RNA-based reprogramming approach. Summary plots and representative TRA-1-60-stained reprogramming wells show the yield of TRA-1-60-positive colonies at day 18 of reprogramming initiated at a plating density of 500 cells per well of a 6-well format dish with two independent human primary neonatal fibroblast lines, FN1 (top) and FN2 (bottom). The reprogramming was accomplished using the indicated numbers of mod-mRNA and m-miRNA transfections performed at either 48 or 72 h intervals using Opti-MEM-8.2 as the transfection buffer. The amount of m-miRNA used per transfection is 20 pmol, and the amount of mod-mRNAs is indicated. Error bars, mean ± s.d. ( n = 3). No statistical difference in the reprogramming efficiencies was observed between 6 and 7 transfections (NS, P > 0.05). P values were calculated using the unpaired two-tailed Student’s t test. Scale bars, 10 mm

    Article Snippet: RNA and RNAiMAX were first diluted in either pH-adjusted Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific) or 1× pH-adjusted PBS as indicated in the corresponding figures. pH-adjusted Opti-MEM or pH-adjusted PBS were used as transfection buffers for complex formation between RNAiMAX and RNA.

    Techniques: Transfection, Staining, Two Tailed Test

    Low initial cell plating densities improve the efficiency of the RNA-based reprogramming approach. a Summary table and representative TRA-1-60-stained reprogramming wells showing the yield of TRA-1-60-positive colonies at day 18 of the optimized RNA-based reprogramming regimen (see Fig. 1b ) initiated with human primary neonatal fibroblasts (FN2) at the indicated cell plating numbers per well of a 6-well format dish. The efficiency was calculated by dividing the number of resulting TRA-1-60-positive colonies by the number of input cells and multiplying by 100%. Mean ± s.d. ( n = 3). Scale bar, 10 mm. Similar results were obtained using an independent primary neonatal fibroblast line, FN1 (Supplementary Fig. 5 ). b Summary table showing the reprogramming of individually plated single cells with the 5fM 3 O mod-mRNA reprogramming cocktail delivered alone or in combination with m-miRNAs every 48 h using Opti-MEM adjusted to a pH of 8.2 as the transfection buffer (see Fig. 1b ). The number of individually plated single cells from three independent primary neonatal fibroblast lines (FN1, FN2, and FN5), the survival of these cells throughout reprogramming, and the resulting number of wells with at least one TRA-1-60-positive colony at day 18 of reprogramming are shown. The efficiency was calculated by dividing the number of wells with TRA-1-60-positive colonies by the number of wells with surviving input cells and multiplying by 100%. Representative TRA-1-60-stained reprogramming wells (48-well dish format) correspond to conditions from the summary table. Note the formation of multiple sister TRA-1-60-positive colonies when both mod-mRNA and m-miRNA transfections were employed. Scale bar, 3 mm. c Representative day-by-day images of single-cell reprogramming (FN2) performed with the optimized RNA-based reprogramming approach. Staining for TRA-1-60 performed on day 18 of reprogramming is also shown. Scale bar, 100 µm

    Journal: Nature Communications

    Article Title: High-efficiency RNA-based reprogramming of human primary fibroblasts

    doi: 10.1038/s41467-018-03190-3

    Figure Lengend Snippet: Low initial cell plating densities improve the efficiency of the RNA-based reprogramming approach. a Summary table and representative TRA-1-60-stained reprogramming wells showing the yield of TRA-1-60-positive colonies at day 18 of the optimized RNA-based reprogramming regimen (see Fig. 1b ) initiated with human primary neonatal fibroblasts (FN2) at the indicated cell plating numbers per well of a 6-well format dish. The efficiency was calculated by dividing the number of resulting TRA-1-60-positive colonies by the number of input cells and multiplying by 100%. Mean ± s.d. ( n = 3). Scale bar, 10 mm. Similar results were obtained using an independent primary neonatal fibroblast line, FN1 (Supplementary Fig. 5 ). b Summary table showing the reprogramming of individually plated single cells with the 5fM 3 O mod-mRNA reprogramming cocktail delivered alone or in combination with m-miRNAs every 48 h using Opti-MEM adjusted to a pH of 8.2 as the transfection buffer (see Fig. 1b ). The number of individually plated single cells from three independent primary neonatal fibroblast lines (FN1, FN2, and FN5), the survival of these cells throughout reprogramming, and the resulting number of wells with at least one TRA-1-60-positive colony at day 18 of reprogramming are shown. The efficiency was calculated by dividing the number of wells with TRA-1-60-positive colonies by the number of wells with surviving input cells and multiplying by 100%. Representative TRA-1-60-stained reprogramming wells (48-well dish format) correspond to conditions from the summary table. Note the formation of multiple sister TRA-1-60-positive colonies when both mod-mRNA and m-miRNA transfections were employed. Scale bar, 3 mm. c Representative day-by-day images of single-cell reprogramming (FN2) performed with the optimized RNA-based reprogramming approach. Staining for TRA-1-60 performed on day 18 of reprogramming is also shown. Scale bar, 100 µm

    Article Snippet: RNA and RNAiMAX were first diluted in either pH-adjusted Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific) or 1× pH-adjusted PBS as indicated in the corresponding figures. pH-adjusted Opti-MEM or pH-adjusted PBS were used as transfection buffers for complex formation between RNAiMAX and RNA.

    Techniques: Staining, Transfection

    Transfection of fluorescent siRNA by SQ-NMe 3 + NPs in A673 human Ewing sarcoma cells. A673 cells were treated for 4 h in serum-free OptiMEM medium by labeled siRNA-FITC either free or bound to SQ-NMe 3 + NPs at a charge ratio (N/P) of 2, 4, or 6 (corresponding to 4, 8, or 12 μM). The cell nucleus was labeled by DAPI. The slides were observed by epifluorescence microscopy. Scale bar is 20 μm.

    Journal: Nucleic Acid Therapeutics

    Article Title: Turning Squalene into Cationic Lipid Allows a Delivery of siRNA in Cultured Cells

    doi: 10.1089/nat.2014.0504

    Figure Lengend Snippet: Transfection of fluorescent siRNA by SQ-NMe 3 + NPs in A673 human Ewing sarcoma cells. A673 cells were treated for 4 h in serum-free OptiMEM medium by labeled siRNA-FITC either free or bound to SQ-NMe 3 + NPs at a charge ratio (N/P) of 2, 4, or 6 (corresponding to 4, 8, or 12 μM). The cell nucleus was labeled by DAPI. The slides were observed by epifluorescence microscopy. Scale bar is 20 μm.

    Article Snippet: Then, the medium was discarded, and 450 μL of OptiMEM serum-free medium (Gibco) was added to the cells.

    Techniques: Transfection, Labeling, Epifluorescence Microscopy

    Transfection of fluorescent siRNA by SQ-NH(NH 2 )C═NH 2 + NPs in A673 human Ewing sarcoma cells. A673 cells were treated for 4 h in serum-free OptiMEM medium by labeled siRNA-FITC ( green ) either free or bound to SQ-NH(NH 2 )C═NH 2 + NPs at a charge ratio (N/P) of 2, 4, or 6 (corresponding to 4, 8, or 12 μM). The cell nucleus was labeled by DAPI ( blue ). The slides were observed by confocal microscopy. Scale bare is 20 μm.

    Journal: Nucleic Acid Therapeutics

    Article Title: Turning Squalene into Cationic Lipid Allows a Delivery of siRNA in Cultured Cells

    doi: 10.1089/nat.2014.0504

    Figure Lengend Snippet: Transfection of fluorescent siRNA by SQ-NH(NH 2 )C═NH 2 + NPs in A673 human Ewing sarcoma cells. A673 cells were treated for 4 h in serum-free OptiMEM medium by labeled siRNA-FITC ( green ) either free or bound to SQ-NH(NH 2 )C═NH 2 + NPs at a charge ratio (N/P) of 2, 4, or 6 (corresponding to 4, 8, or 12 μM). The cell nucleus was labeled by DAPI ( blue ). The slides were observed by confocal microscopy. Scale bare is 20 μm.

    Article Snippet: Then, the medium was discarded, and 450 μL of OptiMEM serum-free medium (Gibco) was added to the cells.

    Techniques: Transfection, Labeling, Confocal Microscopy

    Ultraviolet/visible (UV/vis) absorption spectra of LPEI suspended at varying concentrations in: (a) DI water, or (b) OptiMEM buffer, without siRNA, showed maximum peaks at 240 nm and 244 nm, respectively. (“LPEI concentration (≡ N/P ratio)”

    Journal: Acta biomaterialia

    Article Title: Multilayer mediated forward and patterned siRNA transfection using linear-PEI at extended N/P ratios

    doi: 10.1016/j.actbio.2009.01.004

    Figure Lengend Snippet: Ultraviolet/visible (UV/vis) absorption spectra of LPEI suspended at varying concentrations in: (a) DI water, or (b) OptiMEM buffer, without siRNA, showed maximum peaks at 240 nm and 244 nm, respectively. (“LPEI concentration (≡ N/P ratio)”

    Article Snippet: Lipofectamine 2000 (LF2k) transfection reagent (1 mg ml−1 ), OptiMEM reduced serum medium (catalog no. 31985), Dulbecco’s Modified Eagle Medium (DMEM), penicillin, streptomycin, were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Concentration Assay