lipid based transient transfection  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85

    Structured Review

    Thermo Fisher lipid based transient transfection
    Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and <t>transfections.</t>
    Lipid Based Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid based transient transfection/product/Thermo Fisher
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    lipid based transient transfection - by Bioz Stars, 2020-08
    85/100 stars

    Related Products / Commonly Used Together

    fak
    lipofectamine 2000
    lipofectamine
    mgfp
    cho.k1 cells

    Images

    1) Product Images from "Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains"

    Article Title: Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains

    Journal: ACS chemical biology

    doi: 10.1021/acschembio.7b00732

    Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and transfections.
    Figure Legend Snippet: Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and transfections.

    Techniques Used: Activation Assay, Expressing, Construct, Cycling Probe Technology, Transfection

    Related Articles

    Transfection:

    Article Title: Fluorescence Fluctuation Approaches to the Study of Adhesion and Signaling
    Article Snippet: .. The following is a sample procedure for the lipid-based transient transfection of mGFP–paxillin and FAK–mCherry in CHO.K1 cells using Lipofectamine (Invitrogen) ( ). ..

    Article Title: Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains
    Article Snippet: .. In additional experiments, lipid-based transient transfection was carried out using Lipofectamine 2000 (Thermo-Fisher). .. The human RI α constructs were expressed in E. coli Δcya TP2000 cells, which lack adenylyl cyclase activity., The inoculated liter cultures were incubated at 37 °C with shaking at 180 rpm until growth reaching an optical density of ~0.6 to 0.8, following which 400 μ M IPTG was added to induce protein expression overnight at RT.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Thermo Fisher lipid based transient transfection
    Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and <t>transfections.</t>
    Lipid Based Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid based transient transfection/product/Thermo Fisher
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    lipid based transient transfection - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    99
    Thermo Fisher lipid based transfection
    Copy number analysis. a Copy number profiles of each tumor. Differences in color intensity depend on copy number amplitude and tumor purity. b Broad copy number changes enriched in the metastases ( n = 32) compared with TCGA tumors ( n = 80). Two-tailed Fisher’s exact tests with adjustment for multiple testing using the Benjamini–Hochberg method. c Two primary tumors compared with matched metastases. d Focal deletions of CDKN2A in two samples. e RNA-seq from the UM9 metastasis and corresponding PDX showing the region with focal CDKN2A deletion. f Genes in recurrent arm-level copy number aberrations ranked by associations between gene expression and copy number that were consistent in this cohort and TCGA tumors, and further ranked by protein–protein interaction network degree from the Human Protein Reference Database (HPRD), and additionally by presence of any associations with worse survival. The top three candidates are shown in each region. Connecting lines represent protein interactions of the highest ranked gene per region. Blue represents regions of loss and red regions of gain. Summarized representations of copy number profiles per region show the relative numbers of gain and loss events, with the inner circle representing TCGA samples and the outer our cohort. For the metastatic cohort, n = 28 samples with matching DNA and RNA data were included. g Pathways enriched among the combined set of the top 10 genes per region of gain or loss. h Functional interrogation by siRNA of main candidate genes whose expression is elevated due to copy number alteration. Cells were counted or viability was measured at 72 h, 96 h and 96 h for the cell lines UM22, MP-41, and 92-1, respectively, after <t>transfection</t> of the siRNA pools. n = 3 samples were transfected independently, for each cell line. Data are presented as mean values ± standard error of the mean (SEM). Two-way ANOVA was used to estimate differences, taking into account both cell line and target gene as variables. q values were calculated using Benjamini–Hochberg correction, taking into account all genes in h as well as those in Supplementary Fig. 3c , which shows other candidates of interest investigated. Dotted lines indicate q = 0.05.
    Lipid Based Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid based transfection/product/Thermo Fisher
    Average 99 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    lipid based transfection - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and transfections.

    Journal: ACS chemical biology

    Article Title: Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains

    doi: 10.1021/acschembio.7b00732

    Figure Lengend Snippet: Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and transfections.

    Article Snippet: In additional experiments, lipid-based transient transfection was carried out using Lipofectamine 2000 (Thermo-Fisher).

    Techniques: Activation Assay, Expressing, Construct, Cycling Probe Technology, Transfection

    Copy number analysis. a Copy number profiles of each tumor. Differences in color intensity depend on copy number amplitude and tumor purity. b Broad copy number changes enriched in the metastases ( n = 32) compared with TCGA tumors ( n = 80). Two-tailed Fisher’s exact tests with adjustment for multiple testing using the Benjamini–Hochberg method. c Two primary tumors compared with matched metastases. d Focal deletions of CDKN2A in two samples. e RNA-seq from the UM9 metastasis and corresponding PDX showing the region with focal CDKN2A deletion. f Genes in recurrent arm-level copy number aberrations ranked by associations between gene expression and copy number that were consistent in this cohort and TCGA tumors, and further ranked by protein–protein interaction network degree from the Human Protein Reference Database (HPRD), and additionally by presence of any associations with worse survival. The top three candidates are shown in each region. Connecting lines represent protein interactions of the highest ranked gene per region. Blue represents regions of loss and red regions of gain. Summarized representations of copy number profiles per region show the relative numbers of gain and loss events, with the inner circle representing TCGA samples and the outer our cohort. For the metastatic cohort, n = 28 samples with matching DNA and RNA data were included. g Pathways enriched among the combined set of the top 10 genes per region of gain or loss. h Functional interrogation by siRNA of main candidate genes whose expression is elevated due to copy number alteration. Cells were counted or viability was measured at 72 h, 96 h and 96 h for the cell lines UM22, MP-41, and 92-1, respectively, after transfection of the siRNA pools. n = 3 samples were transfected independently, for each cell line. Data are presented as mean values ± standard error of the mean (SEM). Two-way ANOVA was used to estimate differences, taking into account both cell line and target gene as variables. q values were calculated using Benjamini–Hochberg correction, taking into account all genes in h as well as those in Supplementary Fig. 3c , which shows other candidates of interest investigated. Dotted lines indicate q = 0.05.

    Journal: Nature Communications

    Article Title: Molecular profiling of driver events in metastatic uveal melanoma

    doi: 10.1038/s41467-020-15606-0

    Figure Lengend Snippet: Copy number analysis. a Copy number profiles of each tumor. Differences in color intensity depend on copy number amplitude and tumor purity. b Broad copy number changes enriched in the metastases ( n = 32) compared with TCGA tumors ( n = 80). Two-tailed Fisher’s exact tests with adjustment for multiple testing using the Benjamini–Hochberg method. c Two primary tumors compared with matched metastases. d Focal deletions of CDKN2A in two samples. e RNA-seq from the UM9 metastasis and corresponding PDX showing the region with focal CDKN2A deletion. f Genes in recurrent arm-level copy number aberrations ranked by associations between gene expression and copy number that were consistent in this cohort and TCGA tumors, and further ranked by protein–protein interaction network degree from the Human Protein Reference Database (HPRD), and additionally by presence of any associations with worse survival. The top three candidates are shown in each region. Connecting lines represent protein interactions of the highest ranked gene per region. Blue represents regions of loss and red regions of gain. Summarized representations of copy number profiles per region show the relative numbers of gain and loss events, with the inner circle representing TCGA samples and the outer our cohort. For the metastatic cohort, n = 28 samples with matching DNA and RNA data were included. g Pathways enriched among the combined set of the top 10 genes per region of gain or loss. h Functional interrogation by siRNA of main candidate genes whose expression is elevated due to copy number alteration. Cells were counted or viability was measured at 72 h, 96 h and 96 h for the cell lines UM22, MP-41, and 92-1, respectively, after transfection of the siRNA pools. n = 3 samples were transfected independently, for each cell line. Data are presented as mean values ± standard error of the mean (SEM). Two-way ANOVA was used to estimate differences, taking into account both cell line and target gene as variables. q values were calculated using Benjamini–Hochberg correction, taking into account all genes in h as well as those in Supplementary Fig. 3c , which shows other candidates of interest investigated. Dotted lines indicate q = 0.05.

    Article Snippet: The siRNA duplexes were purchased from Dharmacon (Thermo Fisher Scientific, Waltham, MA, USA) and the lipid based transfection was performed with Lipofectamine-RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) using 1 pmol of siRNA per well of a 96-well plate as per the guidelines provided by manufacturer.

    Techniques: Two Tailed Test, RNA Sequencing Assay, Expressing, Functional Assay, Transfection

    Transcriptional activation and secretion of IFNβ. a Time profiles of Ifnb1 (INFβ) mRNA levels in response to different stimuli. WT MEFs were stimulated with 1000 U/ml IFNβ, 1 μg/ml LPS, or 1 μg/ml poly(I:C) in the absence (left) or presence (right) of IFNAR-blocking antibody (α-IFNAR). Stat1 –/– MEFs and RelA –/– MEFs (right) were also stimulated with 1 μg/ml poly(I:C). α-IFNAR was added to cells at 10 μg/ml at 0, 3, 6 and 10 h after poly(I:C) transfection. Time profiles of relative mRNA levels were obtained with RT-PCR, and then rescaled to absolute numbers using digital PCR measurements (bars represent means ± s.e.m., n ≥ 2; see Supplementary Note for plots of all replicates compared with model simulations). b Secretion of IFNβ in response to TNFα, LPS or poly(I:C). WT MEFs were stimulated with 10 ng/ml TNFα ( n = 2), 1 μg/ml LPS, 0.1, 0.3, 1 and 3 μg/ml poly(I:C), or 1 μg/ml poly(I:C) in the presence of α-IFNAR ( n = 2). Both Stat1 –/– MEFs and RelA –/– MEFs were stimulated with 1 μg/ml or 3 μg/ml poly(I:C). For each condition, IFNβ concentration after 2, 4, 6, 10 and 24 h was measured by ELISA in 200 μl of culture medium harvested from above 25,000 ± 5000 cells. Bars represent means ± s.e.m. ( n )

    Journal: Nature Communications

    Article Title: Cell fate in antiviral response arises in the crosstalk of IRF, NF-κB and JAK/STAT pathways

    doi: 10.1038/s41467-017-02640-8

    Figure Lengend Snippet: Transcriptional activation and secretion of IFNβ. a Time profiles of Ifnb1 (INFβ) mRNA levels in response to different stimuli. WT MEFs were stimulated with 1000 U/ml IFNβ, 1 μg/ml LPS, or 1 μg/ml poly(I:C) in the absence (left) or presence (right) of IFNAR-blocking antibody (α-IFNAR). Stat1 –/– MEFs and RelA –/– MEFs (right) were also stimulated with 1 μg/ml poly(I:C). α-IFNAR was added to cells at 10 μg/ml at 0, 3, 6 and 10 h after poly(I:C) transfection. Time profiles of relative mRNA levels were obtained with RT-PCR, and then rescaled to absolute numbers using digital PCR measurements (bars represent means ± s.e.m., n ≥ 2; see Supplementary Note for plots of all replicates compared with model simulations). b Secretion of IFNβ in response to TNFα, LPS or poly(I:C). WT MEFs were stimulated with 10 ng/ml TNFα ( n = 2), 1 μg/ml LPS, 0.1, 0.3, 1 and 3 μg/ml poly(I:C), or 1 μg/ml poly(I:C) in the presence of α-IFNAR ( n = 2). Both Stat1 –/– MEFs and RelA –/– MEFs were stimulated with 1 μg/ml or 3 μg/ml poly(I:C). For each condition, IFNβ concentration after 2, 4, 6, 10 and 24 h was measured by ELISA in 200 μl of culture medium harvested from above 25,000 ± 5000 cells. Bars represent means ± s.e.m. ( n )

    Article Snippet: Poly(I:C) was delivered to cells by means of lipid-based transfection, using Lipofectamine LTX with Plus Reagent (ThermoFischer Scientific).

    Techniques: Activation Assay, Blocking Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Digital PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of translation stabilises translocation of RelA and IRF3. a RelA (NF-κB) translocation and cytoplasmic IκBα levels in response to LPS or CHX + LPS. WT MEFs were stimulated with 1 μg/ml LPS in the absence or presence of 5 μg/ml CHX, fixed and stained at given time-points with antibodies for RelA and IκBα. Representative excerpt from confocal images show cells at 0 (nt), 30, 90 and 240 min after LPS stimulation. Histograms ( n ≥ 700, from a representative experiment out of 2) show the full time course of RelA nuclear translocation, defined for Fig. 2 . See Supplementary Data 3 for corresponding uncropped immunostaining images. Scale bar: 50 μm. b – d Protein levels in response to poly(I:C) upon PKR inhibition. WT MEFs were stimulated with 1 μg/ml poly(I:C) for 0 (nt), 2 and 4 h in the absence or presence of imidazolo-oxindole PKR inhibitor (1 μM/ml), C16, added at 1 h prior to poly(I:C) transfection. Culture medium for all conditions contained the C16 solvent DMSO (0.5% final concentration), and was FBS-free to prevent interference with C16. b Whole-cell extracts were analysed using antibodies against total PKR, A20 and IκBα, as well as against a phosphorylated (active) form of PKR (p-Thr451). c Nuclear and cytoplasmic fractions were analysed using antibodies against total RelA and IRF3, as well as against their phosphorylated (active) forms, p-RelA (Ser536) and p-IRF3 (Ser396). d Cells were fixed and immunostained for RelA and IRF3. Scatter plots show nuclear translocations of RelA vs. IRF3 ( n = 500) based on confocal images analysis. Percentages indicate fractions of active cells; activity was defined by responding (see also Fig. 2b ) with both RelA and IRF3 translocation, as illustrated in a mock plot at the top. See Supplementary Data 4 for corresponding uncropped immunostaining images

    Journal: Nature Communications

    Article Title: Cell fate in antiviral response arises in the crosstalk of IRF, NF-κB and JAK/STAT pathways

    doi: 10.1038/s41467-017-02640-8

    Figure Lengend Snippet: Inhibition of translation stabilises translocation of RelA and IRF3. a RelA (NF-κB) translocation and cytoplasmic IκBα levels in response to LPS or CHX + LPS. WT MEFs were stimulated with 1 μg/ml LPS in the absence or presence of 5 μg/ml CHX, fixed and stained at given time-points with antibodies for RelA and IκBα. Representative excerpt from confocal images show cells at 0 (nt), 30, 90 and 240 min after LPS stimulation. Histograms ( n ≥ 700, from a representative experiment out of 2) show the full time course of RelA nuclear translocation, defined for Fig. 2 . See Supplementary Data 3 for corresponding uncropped immunostaining images. Scale bar: 50 μm. b – d Protein levels in response to poly(I:C) upon PKR inhibition. WT MEFs were stimulated with 1 μg/ml poly(I:C) for 0 (nt), 2 and 4 h in the absence or presence of imidazolo-oxindole PKR inhibitor (1 μM/ml), C16, added at 1 h prior to poly(I:C) transfection. Culture medium for all conditions contained the C16 solvent DMSO (0.5% final concentration), and was FBS-free to prevent interference with C16. b Whole-cell extracts were analysed using antibodies against total PKR, A20 and IκBα, as well as against a phosphorylated (active) form of PKR (p-Thr451). c Nuclear and cytoplasmic fractions were analysed using antibodies against total RelA and IRF3, as well as against their phosphorylated (active) forms, p-RelA (Ser536) and p-IRF3 (Ser396). d Cells were fixed and immunostained for RelA and IRF3. Scatter plots show nuclear translocations of RelA vs. IRF3 ( n = 500) based on confocal images analysis. Percentages indicate fractions of active cells; activity was defined by responding (see also Fig. 2b ) with both RelA and IRF3 translocation, as illustrated in a mock plot at the top. See Supplementary Data 4 for corresponding uncropped immunostaining images

    Article Snippet: Poly(I:C) was delivered to cells by means of lipid-based transfection, using Lipofectamine LTX with Plus Reagent (ThermoFischer Scientific).

    Techniques: Inhibition, Translocation Assay, Staining, Immunostaining, Transfection, Concentration Assay, Activity Assay

    Transcriptional activation and secretion of IFNβ. a Time profiles of Ifnb1 (INFβ) mRNA levels in response to different stimuli. WT MEFs were stimulated with 1000 U/ml IFNβ, 1 μg/ml LPS, or 1 μg/ml poly(I:C) in the absence (left) or presence (right) of IFNAR-blocking antibody (α-IFNAR). Stat1 –/– MEFs and RelA –/– MEFs (right) were also stimulated with 1 μg/ml poly(I:C). α-IFNAR was added to cells at 10 μg/ml at 0, 3, 6 and 10 h after poly(I:C) transfection. Time profiles of relative mRNA levels were obtained with RT-PCR, and then rescaled to absolute numbers using digital PCR measurements (bars represent means ± s.e.m., n ≥ 2; see Supplementary Note for plots of all replicates compared with model simulations). b Secretion of IFNβ in response to TNFα, LPS or poly(I:C). WT MEFs were stimulated with 10 ng/ml TNFα ( n = 2), 1 μg/ml LPS, 0.1, 0.3, 1 and 3 μg/ml poly(I:C), or 1 μg/ml poly(I:C) in the presence of α-IFNAR ( n = 2). Both Stat1 –/– MEFs and RelA –/– MEFs were stimulated with 1 μg/ml or 3 μg/ml poly(I:C). For each condition, IFNβ concentration after 2, 4, 6, 10 and 24 h was measured by ELISA in 200 μl of culture medium harvested from above 25,000 ± 5000 cells. Bars represent means ± s.e.m. ( n = 3, except where stated otherwise, values of all replicates are provided in Supplementary Table 8 )

    Journal: Nature Communications

    Article Title: Cell fate in antiviral response arises in the crosstalk of IRF, NF-κB and JAK/STAT pathways

    doi: 10.1038/s41467-017-02640-8

    Figure Lengend Snippet: Transcriptional activation and secretion of IFNβ. a Time profiles of Ifnb1 (INFβ) mRNA levels in response to different stimuli. WT MEFs were stimulated with 1000 U/ml IFNβ, 1 μg/ml LPS, or 1 μg/ml poly(I:C) in the absence (left) or presence (right) of IFNAR-blocking antibody (α-IFNAR). Stat1 –/– MEFs and RelA –/– MEFs (right) were also stimulated with 1 μg/ml poly(I:C). α-IFNAR was added to cells at 10 μg/ml at 0, 3, 6 and 10 h after poly(I:C) transfection. Time profiles of relative mRNA levels were obtained with RT-PCR, and then rescaled to absolute numbers using digital PCR measurements (bars represent means ± s.e.m., n ≥ 2; see Supplementary Note for plots of all replicates compared with model simulations). b Secretion of IFNβ in response to TNFα, LPS or poly(I:C). WT MEFs were stimulated with 10 ng/ml TNFα ( n = 2), 1 μg/ml LPS, 0.1, 0.3, 1 and 3 μg/ml poly(I:C), or 1 μg/ml poly(I:C) in the presence of α-IFNAR ( n = 2). Both Stat1 –/– MEFs and RelA –/– MEFs were stimulated with 1 μg/ml or 3 μg/ml poly(I:C). For each condition, IFNβ concentration after 2, 4, 6, 10 and 24 h was measured by ELISA in 200 μl of culture medium harvested from above 25,000 ± 5000 cells. Bars represent means ± s.e.m. ( n = 3, except where stated otherwise, values of all replicates are provided in Supplementary Table 8 )

    Article Snippet: Poly(I:C) was delivered to cells by means of lipid-based transfection, using Lipofectamine LTX with Plus Reagent (ThermoFischer Scientific).

    Techniques: Activation Assay, Blocking Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Digital PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay