transfection mixture  (Thermo Fisher)


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    Name:
    Lipofectamine Transfection Reagent
    Description:
    Lipofectamine Transfection Reagent is one of our first generation cationic lipid transfection reagents formulated for the transfection of DNA into eukaryotic cells Lipofectamine Transfection Reagent is a trusted value reagent with many years of cited use in thousands of publications and with many cell lines With Lipofectamine Transfection Reagent you ll get • A reagent proven to work in high throughput applications• A reliable reagent for establishing stable cell lines• A reagent that works well with PLUS Reagent for higher protein expressionA proven technologyOur Lipofectamine brand reagents have been recognized as the most cited transfection reagent family with tens of thousands of citations since launch in 1993 Lipofectamine Transfection Reagent is no exception as a trusted reagent for a wide range of cell lines When used in combination with PLUS Reagent Lipofectamine Transfection Reagent has been shown to work well with cells such as BHK 21 NIH 3T3 COS 1 fibroblasts keratinocytes HT 29 MRC 5 and SK BR3 A value alternative for plasmid DNAWith such a legacy comes a commitment to providing the best reagents for all scientists and labs regardless of their funding capabilities Lipofectamine Transfection Reagent is a value alternative for transfection of plasmid DNA into eukaryotic cells with a simplified protocol For optimal results in a wider range of plasmid DNA transfection conditions we recommend Lipofectamine LTX Reagent due to its optimal balance of potency low cytotoxicity Lipofectamine 2000 Transfection Reagent is recommended if you are transfecting a broad range of cell lines or want to transfect RNA
    Catalog Number:
    18324010
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Plasmid Transfection|Stem Cell & Primary Cell Transfections|Transfection
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    Structured Review

    Thermo Fisher transfection mixture
    Plasma membrane distribution of M-PMV Env. The COS-1 cells were either transfected with plasmids encoding M-PMV genomic DNA with: WT Env (pSARMX-WT; panel A ), I18A Env (pSARMX-I18A; panel B ), or Y22A Env (pSARMX-Y22A; panel C ) or an M-PMV Env expression vector (pTMT-WT; panel D ). 48 h <t>post-transfection</t> cells were incubated with goat anti-M-PMV antibody on ice for 25 min to bind surface exposed M-PMV Env. Formaldehyde (4%) fixed cells were immunostained for CA protein with rabbit anti-CA antibody (except for the cells transfected with pTMT-WT,). Env was visualized using secondary anti-goat IgG antibody conjugated with Alexa Fluor ® 350. Samples were mounted in Vectashield mounting media and imaged on an Olympus cell^R microscope. The original blue color of the AF350 signal was changed to cyan for increased contrast. Magnification 600×; scale bars 20 µm.
    Lipofectamine Transfection Reagent is one of our first generation cationic lipid transfection reagents formulated for the transfection of DNA into eukaryotic cells Lipofectamine Transfection Reagent is a trusted value reagent with many years of cited use in thousands of publications and with many cell lines With Lipofectamine Transfection Reagent you ll get • A reagent proven to work in high throughput applications• A reliable reagent for establishing stable cell lines• A reagent that works well with PLUS Reagent for higher protein expressionA proven technologyOur Lipofectamine brand reagents have been recognized as the most cited transfection reagent family with tens of thousands of citations since launch in 1993 Lipofectamine Transfection Reagent is no exception as a trusted reagent for a wide range of cell lines When used in combination with PLUS Reagent Lipofectamine Transfection Reagent has been shown to work well with cells such as BHK 21 NIH 3T3 COS 1 fibroblasts keratinocytes HT 29 MRC 5 and SK BR3 A value alternative for plasmid DNAWith such a legacy comes a commitment to providing the best reagents for all scientists and labs regardless of their funding capabilities Lipofectamine Transfection Reagent is a value alternative for transfection of plasmid DNA into eukaryotic cells with a simplified protocol For optimal results in a wider range of plasmid DNA transfection conditions we recommend Lipofectamine LTX Reagent due to its optimal balance of potency low cytotoxicity Lipofectamine 2000 Transfection Reagent is recommended if you are transfecting a broad range of cell lines or want to transfect RNA
    https://www.bioz.com/result/transfection mixture/product/Thermo Fisher
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    Images

    1) Product Images from "Mason-Pfizer Monkey Virus Envelope Glycoprotein Cycling and Its Vesicular Co-Transport with Immature Particles"

    Article Title: Mason-Pfizer Monkey Virus Envelope Glycoprotein Cycling and Its Vesicular Co-Transport with Immature Particles

    Journal: Viruses

    doi: 10.3390/v10100575

    Plasma membrane distribution of M-PMV Env. The COS-1 cells were either transfected with plasmids encoding M-PMV genomic DNA with: WT Env (pSARMX-WT; panel A ), I18A Env (pSARMX-I18A; panel B ), or Y22A Env (pSARMX-Y22A; panel C ) or an M-PMV Env expression vector (pTMT-WT; panel D ). 48 h post-transfection cells were incubated with goat anti-M-PMV antibody on ice for 25 min to bind surface exposed M-PMV Env. Formaldehyde (4%) fixed cells were immunostained for CA protein with rabbit anti-CA antibody (except for the cells transfected with pTMT-WT,). Env was visualized using secondary anti-goat IgG antibody conjugated with Alexa Fluor ® 350. Samples were mounted in Vectashield mounting media and imaged on an Olympus cell^R microscope. The original blue color of the AF350 signal was changed to cyan for increased contrast. Magnification 600×; scale bars 20 µm.
    Figure Legend Snippet: Plasma membrane distribution of M-PMV Env. The COS-1 cells were either transfected with plasmids encoding M-PMV genomic DNA with: WT Env (pSARMX-WT; panel A ), I18A Env (pSARMX-I18A; panel B ), or Y22A Env (pSARMX-Y22A; panel C ) or an M-PMV Env expression vector (pTMT-WT; panel D ). 48 h post-transfection cells were incubated with goat anti-M-PMV antibody on ice for 25 min to bind surface exposed M-PMV Env. Formaldehyde (4%) fixed cells were immunostained for CA protein with rabbit anti-CA antibody (except for the cells transfected with pTMT-WT,). Env was visualized using secondary anti-goat IgG antibody conjugated with Alexa Fluor ® 350. Samples were mounted in Vectashield mounting media and imaged on an Olympus cell^R microscope. The original blue color of the AF350 signal was changed to cyan for increased contrast. Magnification 600×; scale bars 20 µm.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Incubation, Microscopy

    Rab-markers based identification of intracellular vesicles carrying mCherryTM protein variants. The COS-1 cells co-transfected with a combination of pSARMXmCherryTM and enhanced green fluorescent protein (EGFP)-tagged endosomal marker coding plasmids were fixed with 4% formaldehyde 24 h post-transfection. Upon mounting in Vectashield media the samples were imaged using an Olympus cell^R microscope. White arrowheads indicate colocalization of the mCherryTM signal and the corresponding marker. Magnification 600×; scale bars 20 µm.
    Figure Legend Snippet: Rab-markers based identification of intracellular vesicles carrying mCherryTM protein variants. The COS-1 cells co-transfected with a combination of pSARMXmCherryTM and enhanced green fluorescent protein (EGFP)-tagged endosomal marker coding plasmids were fixed with 4% formaldehyde 24 h post-transfection. Upon mounting in Vectashield media the samples were imaged using an Olympus cell^R microscope. White arrowheads indicate colocalization of the mCherryTM signal and the corresponding marker. Magnification 600×; scale bars 20 µm.

    Techniques Used: Transfection, Marker, Microscopy

    2) Product Images from "Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis"

    Article Title: Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.10.001

    RICTOR depletion in human and murine melanoma cell lines and its impact on growth and motility in vitro . (A, B) Upon transient transfection with two different siRNAs (RICTOR si1, RICTOR si2), RICTOR expression is impaired compared to controls (ctrl. Si = scramble RNA), as determined by Western blotting (A) and densitometry (B). (C) In B16 cells, significant reduction of MTT signal was detected after 24 hours. After 48 hours, a growth inhibitory effect is only observed upon RICTOR si1 ( # ,* P
    Figure Legend Snippet: RICTOR depletion in human and murine melanoma cell lines and its impact on growth and motility in vitro . (A, B) Upon transient transfection with two different siRNAs (RICTOR si1, RICTOR si2), RICTOR expression is impaired compared to controls (ctrl. Si = scramble RNA), as determined by Western blotting (A) and densitometry (B). (C) In B16 cells, significant reduction of MTT signal was detected after 24 hours. After 48 hours, a growth inhibitory effect is only observed upon RICTOR si1 ( # ,* P

    Techniques Used: In Vitro, Transfection, Expressing, Western Blot, MTT Assay

    3) Product Images from "The Toca-1-N-WASP Complex Links Filopodial Formation to Endocytosis S⃞"

    Article Title: The Toca-1-N-WASP Complex Links Filopodial Formation to Endocytosis S⃞

    Journal:

    doi: 10.1074/jbc.M805940200

    N-WASP-induced filopodial formation requires and synergizes with Toca-1. A , N1E115 cells were transfected with N-WASP alone, N-WASP with Toca-1 mutants, and N-WASP with Toca-1 ( a ). All transfections were carried out with GFP-actin. Panels show GFP
    Figure Legend Snippet: N-WASP-induced filopodial formation requires and synergizes with Toca-1. A , N1E115 cells were transfected with N-WASP alone, N-WASP with Toca-1 mutants, and N-WASP with Toca-1 ( a ). All transfections were carried out with GFP-actin. Panels show GFP

    Techniques Used: Transfection

    4) Product Images from "Correction of a Factor VIII genomic inversion with designer-recombinases"

    Article Title: Correction of a Factor VIII genomic inversion with designer-recombinases

    Journal: bioRxiv

    doi: 10.1101/2020.11.02.328013

    Activity of the D7 heterodimer in human cells. a) Schematic overview of the integrated reporter construct in HEK293T cells. Site-specific recombination excises the spleen focus forming virus (SFFV) promoter-driven puromycin resistance gene (PURO), which leads to expression of the red-fluorescent mCherry gene. b) Depiction of the mRNAs that were transfected for transient expression of the recombinase dimer. tagBFP mRNA was used to monitor transfection efficiencies. NLS, nuclear localization signal. c) Fluorescent and brightfield images of transfected HEK293T loxF8 reporter cells with indicated recombinases. Note that mCherry expression is only visible in cells receiving both monomers. 200 μm white scale bars are indicated. d) Quantification of the recombination efficiencies 48 h after transfection of HEK293T loxF8 reporter cells with indicated recombinases, analyzed by FACS (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Recombination efficiencies were calculated based on the presented formula. e) Schematic overview of a fraction of the F8 gene displaying the PCR primers used to detect the orientation of the loxF8 locus. Exons are displayed in magenta and the repeated regions int1h-1 and int1h-2 are shown in grey. Primer binding sites are indicated with arrows. The transcription start site is depicted by a black arrow. f) Gel image of PCR products generated using indicated primer combinations to detect the orientation of the loxF8 locus with and without treatment with the D7 heterodimer. Band sizes of the marker lane (M) are indicated.
    Figure Legend Snippet: Activity of the D7 heterodimer in human cells. a) Schematic overview of the integrated reporter construct in HEK293T cells. Site-specific recombination excises the spleen focus forming virus (SFFV) promoter-driven puromycin resistance gene (PURO), which leads to expression of the red-fluorescent mCherry gene. b) Depiction of the mRNAs that were transfected for transient expression of the recombinase dimer. tagBFP mRNA was used to monitor transfection efficiencies. NLS, nuclear localization signal. c) Fluorescent and brightfield images of transfected HEK293T loxF8 reporter cells with indicated recombinases. Note that mCherry expression is only visible in cells receiving both monomers. 200 μm white scale bars are indicated. d) Quantification of the recombination efficiencies 48 h after transfection of HEK293T loxF8 reporter cells with indicated recombinases, analyzed by FACS (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Recombination efficiencies were calculated based on the presented formula. e) Schematic overview of a fraction of the F8 gene displaying the PCR primers used to detect the orientation of the loxF8 locus. Exons are displayed in magenta and the repeated regions int1h-1 and int1h-2 are shown in grey. Primer binding sites are indicated with arrows. The transcription start site is depicted by a black arrow. f) Gel image of PCR products generated using indicated primer combinations to detect the orientation of the loxF8 locus with and without treatment with the D7 heterodimer. Band sizes of the marker lane (M) are indicated.

    Techniques Used: Activity Assay, Construct, Expressing, Transfection, FACS, Standard Deviation, Polymerase Chain Reaction, Binding Assay, Generated, Marker

    Strategy to evaluate recombinases in mammalian cells. a) Schematic representation of the generation of the HEK293T loxF8 reporter cell line. Important steps are shown on the time-line. b) Representative FACS plot and gating strategy to evaluate the transfection efficiency measured by tagBFP+ cells (transfection of tagBFP mRNA only). c) Representative FACS plot and gating strategy of HEK293T loxF8 reporter cells that were transfected with tagBFP and indicated recombinase mRNAs. Upon successful site-specific recombination, cells express mCherry. tagBFP+ and mCherry+ were used to calculate the recombination efficiencies (see formula in Fig. 3 ). Parts of the figure are created with BioRender.com .
    Figure Legend Snippet: Strategy to evaluate recombinases in mammalian cells. a) Schematic representation of the generation of the HEK293T loxF8 reporter cell line. Important steps are shown on the time-line. b) Representative FACS plot and gating strategy to evaluate the transfection efficiency measured by tagBFP+ cells (transfection of tagBFP mRNA only). c) Representative FACS plot and gating strategy of HEK293T loxF8 reporter cells that were transfected with tagBFP and indicated recombinase mRNAs. Upon successful site-specific recombination, cells express mCherry. tagBFP+ and mCherry+ were used to calculate the recombination efficiencies (see formula in Fig. 3 ). Parts of the figure are created with BioRender.com .

    Techniques Used: FACS, Transfection

    Functional correction of the inverted F8 gene in differentiated endothelial cells. a) Schematic overview of the EC differentiation protocol. Important steps are highlighted on the timeline. b) Efficiency of transfection of mCherry mRNA to differentiated endothelial cells. Brightfield (BF) image, mCherry image and the merged image a shown. Images were taken 48 h post transfection. White bars indicated 50 μm scale bar. c) RecF8 expression corrects the int1h inversion. The arrangement of different primers around the first and second loxF8 site to detect the orientation of the full 140 kb fragment before and after RecF8 treatment are shown in the top panels. The lower panels show gels of PCR products obtained on genomic DNA from patient specific ECs with and without treatment with RecF8. The combination of the primers used for every PCR gel picture is shown below. d) qPCR-based quantification of the genomic inversion efficiencies in ECs after RecF8 treatment. The quantification of the inversion was extrapolated from a standard of defined ratios of WT and F8 patient iPSCs genomic DNA (1%, 5%, 10%, 25% and 50% normal orientation of the loxF8 locus, n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). e) qPCR-based quantification of Factor VIII mRNA transcript (amplification of exon1-exon2 boundary transcript). The relative expression of the Factor VIII mRNA was measured after treating patient specific ECs with indicated amounts of RecF8 mRNA (n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Factor VIII mRNA expression in untreated WT ECs was used for normalization. The Factor VIII mRNA expression was normalized against ß-actin expression. f) Sequencing read of Factor VIII cDNA of RecF8 treated patient-derived ECs. Note that the exon1-exon2 boundary can only occur, if the inversion is corrected to the WT orientation, the gene is transcribed and the pre-mRNA spliced correctly. Parts of the figure were created with BioRender.com .
    Figure Legend Snippet: Functional correction of the inverted F8 gene in differentiated endothelial cells. a) Schematic overview of the EC differentiation protocol. Important steps are highlighted on the timeline. b) Efficiency of transfection of mCherry mRNA to differentiated endothelial cells. Brightfield (BF) image, mCherry image and the merged image a shown. Images were taken 48 h post transfection. White bars indicated 50 μm scale bar. c) RecF8 expression corrects the int1h inversion. The arrangement of different primers around the first and second loxF8 site to detect the orientation of the full 140 kb fragment before and after RecF8 treatment are shown in the top panels. The lower panels show gels of PCR products obtained on genomic DNA from patient specific ECs with and without treatment with RecF8. The combination of the primers used for every PCR gel picture is shown below. d) qPCR-based quantification of the genomic inversion efficiencies in ECs after RecF8 treatment. The quantification of the inversion was extrapolated from a standard of defined ratios of WT and F8 patient iPSCs genomic DNA (1%, 5%, 10%, 25% and 50% normal orientation of the loxF8 locus, n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). e) qPCR-based quantification of Factor VIII mRNA transcript (amplification of exon1-exon2 boundary transcript). The relative expression of the Factor VIII mRNA was measured after treating patient specific ECs with indicated amounts of RecF8 mRNA (n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Factor VIII mRNA expression in untreated WT ECs was used for normalization. The Factor VIII mRNA expression was normalized against ß-actin expression. f) Sequencing read of Factor VIII cDNA of RecF8 treated patient-derived ECs. Note that the exon1-exon2 boundary can only occur, if the inversion is corrected to the WT orientation, the gene is transcribed and the pre-mRNA spliced correctly. Parts of the figure were created with BioRender.com .

    Techniques Used: Functional Assay, Transfection, Expressing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation, Amplification, Sequencing, Derivative Assay

    RecF8 induces the genomic inversion of the loxF8 locus in mammalian cells. a) qPCR-based quantification of the genomic inversion efficiencies at the loxF8 locus after treatment with indicated recombinases in HEK293T cells (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). The quantification of the inversion was extrapolated from a standard of defined ratios of WT and F8 patient iPSCs genomic DNA (1%, 5%, 10%, 25% and 50% hemophilic orientation of the loxF8 locus, n=3). b) Transfection of patient derived induced pluripotent stem cells (iPSCs) with NLS-EGFP mRNA. A representative microscopy image of 48 h post transfection with 1 μg of NLS-EGFP mRNA is shown. White bar indicates 200 μm scale bar. A FACS histogram of mock and NLS-EGFP transfected iPSCs 48 h post transfection is shown to the right. NLS, nuclear localization sequence. c) PCR-based detection of loxF8 locus inversion in WT and patient derived iPSCs after RecF8 treatment. Marker (M) lanes at 1 kb and 3 kb are indicated. Sequencing reads of the PCR product confirming the corrected orientation of the DNA sequence after RecF8 treatment are displayed. The black and grey arrows indicate the sequence orientation between the int1h-1 and int1h-2 repeats of the loxF8 locus.
    Figure Legend Snippet: RecF8 induces the genomic inversion of the loxF8 locus in mammalian cells. a) qPCR-based quantification of the genomic inversion efficiencies at the loxF8 locus after treatment with indicated recombinases in HEK293T cells (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). The quantification of the inversion was extrapolated from a standard of defined ratios of WT and F8 patient iPSCs genomic DNA (1%, 5%, 10%, 25% and 50% hemophilic orientation of the loxF8 locus, n=3). b) Transfection of patient derived induced pluripotent stem cells (iPSCs) with NLS-EGFP mRNA. A representative microscopy image of 48 h post transfection with 1 μg of NLS-EGFP mRNA is shown. White bar indicates 200 μm scale bar. A FACS histogram of mock and NLS-EGFP transfected iPSCs 48 h post transfection is shown to the right. NLS, nuclear localization sequence. c) PCR-based detection of loxF8 locus inversion in WT and patient derived iPSCs after RecF8 treatment. Marker (M) lanes at 1 kb and 3 kb are indicated. Sequencing reads of the PCR product confirming the corrected orientation of the DNA sequence after RecF8 treatment are displayed. The black and grey arrows indicate the sequence orientation between the int1h-1 and int1h-2 repeats of the loxF8 locus.

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation, Transfection, Derivative Assay, Microscopy, FACS, Sequencing, Polymerase Chain Reaction, Marker

    5) Product Images from "KLF4 Acts as a wt-CFTR Suppressor through an AKT-Mediated Pathway"

    Article Title: KLF4 Acts as a wt-CFTR Suppressor through an AKT-Mediated Pathway

    Journal: Cells

    doi: 10.3390/cells9071607

    KLF4 overexpression caused a downregulation of wt-CFTR expression and function. ( A ) Transfection with KLF4-GFP was performed, and the effects of KLF4 overexpression on CFTR expression were assessed by WB. Representative WB of KLF4 and CFTR expression in wt- and F508del–CFTR CFBE cells transfected with either negative control (+GFP) or KLF4-GFP (+KLF4-GFP). Beta-tubulin was used as loading control. Data are normalized to loading control and shown as relative expression (vs. wt-CFTR (+GFP)). (n = 3, unpaired t -test, p
    Figure Legend Snippet: KLF4 overexpression caused a downregulation of wt-CFTR expression and function. ( A ) Transfection with KLF4-GFP was performed, and the effects of KLF4 overexpression on CFTR expression were assessed by WB. Representative WB of KLF4 and CFTR expression in wt- and F508del–CFTR CFBE cells transfected with either negative control (+GFP) or KLF4-GFP (+KLF4-GFP). Beta-tubulin was used as loading control. Data are normalized to loading control and shown as relative expression (vs. wt-CFTR (+GFP)). (n = 3, unpaired t -test, p

    Techniques Used: Over Expression, Expressing, Transfection, Western Blot, Negative Control

    6) Product Images from "LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State"

    Article Title: LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00118

    Meprin β cleaves sAPP dimers with a higher affinity than monomeric sAPP . HEK 293T cells were transiently transfected with the APP695 wt or the APP695 K587C construct either alone or in co-transfection with meprin β. (A) Conditioned medium was probed 24 h post transfection with the antibody mix 1G75A3 (1:3,000) directed against the APP ectodomain. Meprin β expression resulted in a reduced signal intensity of soluble APP, especially prominent for sAPP dimers in cells expressing the APP Cys-mutant (APP695 K587C). (B) For quantification the ratio of sAPP monomers and sAPP dimers, respectively, was calculated as the quotient of signal intensities in cells just transfected with an APP construct (w/o meprin β) to appropriate cells co-expressing meprin β (with meprin β). This analysis revealed a similar reduction in dimerized and monomeric sAPP for cells transfected with APP695 wt. In contrast, HEK cells expressing the dimer bearing construct show a significant increase in the sAPP dimer ratio compared to sAPP monomer ratio. Bars represent mean values ± SEM, n = 4; one-way ANOVA with Tukey's post-hoc test; p
    Figure Legend Snippet: Meprin β cleaves sAPP dimers with a higher affinity than monomeric sAPP . HEK 293T cells were transiently transfected with the APP695 wt or the APP695 K587C construct either alone or in co-transfection with meprin β. (A) Conditioned medium was probed 24 h post transfection with the antibody mix 1G75A3 (1:3,000) directed against the APP ectodomain. Meprin β expression resulted in a reduced signal intensity of soluble APP, especially prominent for sAPP dimers in cells expressing the APP Cys-mutant (APP695 K587C). (B) For quantification the ratio of sAPP monomers and sAPP dimers, respectively, was calculated as the quotient of signal intensities in cells just transfected with an APP construct (w/o meprin β) to appropriate cells co-expressing meprin β (with meprin β). This analysis revealed a similar reduction in dimerized and monomeric sAPP for cells transfected with APP695 wt. In contrast, HEK cells expressing the dimer bearing construct show a significant increase in the sAPP dimer ratio compared to sAPP monomer ratio. Bars represent mean values ± SEM, n = 4; one-way ANOVA with Tukey's post-hoc test; p

    Techniques Used: Transfection, Construct, Cotransfection, Expressing, Mutagenesis

    Faster trafficking of SDS- and heat-stable APP cis -dimers in LRP1-deficient cells. (A) Schematic representation of the APP Cys-mutant encoded by the pLHCX-APP695 K587C construct and its resulting cis -dimerization state. (B,C) CHO K1 cells were transiently transfected with the APP695 K587C construct. (B) 24 h post transfection monomeric as well as dimerized APP were detected by the antibody mix 1G75A3 (1:3,000) in cell lysates. (B,C) Heating of samples up to 95°C shows a negligible reduction of the APP dimer signal with a comparable increase in monomeric APP. (D) CHO K1 and LRP1-deficient CHO 13-5-1 cells both stably expressing the dimer bearing APP construct were pulsed with radiolabeled sulfur ( 35 S) for 15 min. Chase was performed after stated time spans prior to immunoprecipitation of APP with the antibody mix 1G75A3 (1:300) and SDS-PAGE. Exposure of the film revealed an earlier occurrence of soluble APP dimers in the conditioned medium of LRP1-deficient CHO 13-5-1 cells (30 min chase) than in CHO K1 cells (1 h chase). (E) Comparison of the sAPP dimer to APP dimer ratio of both cell types shows a significant elevation for CHO 13-5-1 cells beginning after a 1 h chase. Bars represent mean values ± SEM, n = 3; Student's t -test; p
    Figure Legend Snippet: Faster trafficking of SDS- and heat-stable APP cis -dimers in LRP1-deficient cells. (A) Schematic representation of the APP Cys-mutant encoded by the pLHCX-APP695 K587C construct and its resulting cis -dimerization state. (B,C) CHO K1 cells were transiently transfected with the APP695 K587C construct. (B) 24 h post transfection monomeric as well as dimerized APP were detected by the antibody mix 1G75A3 (1:3,000) in cell lysates. (B,C) Heating of samples up to 95°C shows a negligible reduction of the APP dimer signal with a comparable increase in monomeric APP. (D) CHO K1 and LRP1-deficient CHO 13-5-1 cells both stably expressing the dimer bearing APP construct were pulsed with radiolabeled sulfur ( 35 S) for 15 min. Chase was performed after stated time spans prior to immunoprecipitation of APP with the antibody mix 1G75A3 (1:300) and SDS-PAGE. Exposure of the film revealed an earlier occurrence of soluble APP dimers in the conditioned medium of LRP1-deficient CHO 13-5-1 cells (30 min chase) than in CHO K1 cells (1 h chase). (E) Comparison of the sAPP dimer to APP dimer ratio of both cell types shows a significant elevation for CHO 13-5-1 cells beginning after a 1 h chase. Bars represent mean values ± SEM, n = 3; Student's t -test; p

    Techniques Used: Mutagenesis, Construct, Transfection, Stable Transfection, Expressing, Immunoprecipitation, SDS Page

    LRP1 recruits APP in common transport vesicles . Primary hippocampal neurons from mice (P0) were differentiated for 6 days in vitro , expressing either (A) only LRP1-GFP, (B) APP-RFP or (E,F) alternatively co-expressing both, APP-RFP and LRP1-GFP were subjected for live cell imaging 18–20 h post transfection. Time lapse series were plotted as kymographs (representative kymographs, single transfection: A , B ; co-transfection: E , F ) and used for determination of individual transport vesicle velocities. For quantification of transport velocities > 5 kymographs from different cells were analyzed [ (C,D) LRP: n = 5 cells, n = 534 vesicles; APP: n = 7 cells, n = 254 vesicles; (G,H) n = 5 cells, n = 371 vesicles]. (C) Anterograde and (D) retrograde transport vesicles containing APP-RFP (white columns) or LRP-GFP (black columns). Note the change of APP-RFP transport characteristcs in (G) anterograde and (H) retrograde direction (light gray columns) upon co-expression of (G,H) LRP1-GFP (dark gray columns). Bars represent mean values ± SEM, n > 5 (≥254 vesicles); Student's t -test, p
    Figure Legend Snippet: LRP1 recruits APP in common transport vesicles . Primary hippocampal neurons from mice (P0) were differentiated for 6 days in vitro , expressing either (A) only LRP1-GFP, (B) APP-RFP or (E,F) alternatively co-expressing both, APP-RFP and LRP1-GFP were subjected for live cell imaging 18–20 h post transfection. Time lapse series were plotted as kymographs (representative kymographs, single transfection: A , B ; co-transfection: E , F ) and used for determination of individual transport vesicle velocities. For quantification of transport velocities > 5 kymographs from different cells were analyzed [ (C,D) LRP: n = 5 cells, n = 534 vesicles; APP: n = 7 cells, n = 254 vesicles; (G,H) n = 5 cells, n = 371 vesicles]. (C) Anterograde and (D) retrograde transport vesicles containing APP-RFP (white columns) or LRP-GFP (black columns). Note the change of APP-RFP transport characteristcs in (G) anterograde and (H) retrograde direction (light gray columns) upon co-expression of (G,H) LRP1-GFP (dark gray columns). Bars represent mean values ± SEM, n > 5 (≥254 vesicles); Student's t -test, p

    Techniques Used: Mouse Assay, In Vitro, Expressing, Live Cell Imaging, Transfection, Cotransfection

    Reduced internalization of APP dimers leads to increased sAPP dimer generation . Lysates and conditioned media were probed (24 h post transfection, B ) with antibodies specific for APP (1G75A3 antibody mix, 1:3,000) or actin (1:1,000). LRP1-deficiency in CHO cells (A) stably or (B) transiently transfected with pLHCX-APP695 K587C lead to increased sAPP dimer production. (A) The ratio of sAPP dimers to APP dimers is significantly increased in CHO 13-5-1 cells ( n = 7) compared to CHO K1 cells ( n = 5). Bars represent mean values ± SEM; Student's t -test; p
    Figure Legend Snippet: Reduced internalization of APP dimers leads to increased sAPP dimer generation . Lysates and conditioned media were probed (24 h post transfection, B ) with antibodies specific for APP (1G75A3 antibody mix, 1:3,000) or actin (1:1,000). LRP1-deficiency in CHO cells (A) stably or (B) transiently transfected with pLHCX-APP695 K587C lead to increased sAPP dimer production. (A) The ratio of sAPP dimers to APP dimers is significantly increased in CHO 13-5-1 cells ( n = 7) compared to CHO K1 cells ( n = 5). Bars represent mean values ± SEM; Student's t -test; p

    Techniques Used: Transfection, Stable Transfection

    APP dimer generation and processing takes place in primary cortical neurons. (A) Murine primary cortical neurons (DIV 7) were infected with an adenoviral vector encoding human APP695 while (B) HEK 293T cells were transiently transfected with the pLHCX-APP695 wt construct. 24 h post infection or transfection, respectively, conditioned media (CM) were collected and cells were lysed in RIPA (PCN) or NP-40 (HEK) lysis buffer. Via the antibody mix 1G75A3 (1:3,000) APP was detected in lysates (upper blots) and conditioned media (lower blots). PCN show similar APP dimer expression in the lysate as HEK cells and also generate soluble APP dimers. Under reducing conditions using β-mercaptoethanol (βME) and heating at 95°C the dimer band disappeared. All lanes of lysate or conditioned medium are on the same blot but were rearranged for better presentation.
    Figure Legend Snippet: APP dimer generation and processing takes place in primary cortical neurons. (A) Murine primary cortical neurons (DIV 7) were infected with an adenoviral vector encoding human APP695 while (B) HEK 293T cells were transiently transfected with the pLHCX-APP695 wt construct. 24 h post infection or transfection, respectively, conditioned media (CM) were collected and cells were lysed in RIPA (PCN) or NP-40 (HEK) lysis buffer. Via the antibody mix 1G75A3 (1:3,000) APP was detected in lysates (upper blots) and conditioned media (lower blots). PCN show similar APP dimer expression in the lysate as HEK cells and also generate soluble APP dimers. Under reducing conditions using β-mercaptoethanol (βME) and heating at 95°C the dimer band disappeared. All lanes of lysate or conditioned medium are on the same blot but were rearranged for better presentation.

    Techniques Used: Infection, Plasmid Preparation, Transfection, Construct, Lysis, Expressing

    7) Product Images from "Splicing factors PTBP1 and PTBP2 promote proliferation and migration of glioma cell lines"

    Article Title: Splicing factors PTBP1 and PTBP2 promote proliferation and migration of glioma cell lines

    Journal: Brain

    doi: 10.1093/brain/awp153

    Ectopic overexpression of Nogo-A slows U251 cell proliferation. Cell proliferation curves were performed on U251 cells following transfection and pooled selection with either empty vector or plasmid specifically expressing Nogo-A. Growth was monitored
    Figure Legend Snippet: Ectopic overexpression of Nogo-A slows U251 cell proliferation. Cell proliferation curves were performed on U251 cells following transfection and pooled selection with either empty vector or plasmid specifically expressing Nogo-A. Growth was monitored

    Techniques Used: Over Expression, Transfection, Selection, Plasmid Preparation, Expressing

    8) Product Images from "Distinct temporal expression of GW182 in neurons regulates dendritic arborization"

    Article Title: Distinct temporal expression of GW182 in neurons regulates dendritic arborization

    Journal: bioRxiv

    doi: 10.1101/2020.12.05.412932

    Overexpression of DNGW182 mutant decreases dendritic arborization during a distinct neurodevelopmental window. A) Schematic showing the experimental procedure and timeline: Cultured hippocampal neurons were transfected with either control GFP or GFP DNGW182 on DIV3 and fixed on DIV7, followed by immunostaining. Dendrites were identified with MAP2 staining. B) Representative micrographs of DIV7 cultured hippocampal neurons transfected with either control GFP or GFP DNGW182. The images are derived from the threshold MAP2 intensity of transfected neurons. Scale bar represents 50 microns. C) Intersection profile / Sholl curve of DIV7 cultured hippocampal neurons transfected with either control GFP or GFP DNGW182. Data: mean +/- SEM, n=32 neurons from 4 independent experiments, GFP DNGW182 overexpressing neurons had significantly more dendrites from GFP overexpressing neurons at 16-48 microns from the soma, Two way ANOVA followed by Bonferroni’s multiple comparisons test. D) Quantification of the total dendritic length of cultured hippocampal neurons transfected with either control GFP vector or DN GW182, n=27-32 neurons from 4 independent cultures, Mann-Whitney test. E) Schematic showing the experimental procedure and timeline: Cultured hippocampal neurons were transfected with either control GFP or GFP DNGW182 on DIV7 and fixed on DIV11, followed by immunostaining. Dendrites were identified with MAP2 staining F) Representative micrographs of DIV11 cultured hippocampal neurons transfected with either control GFP vector or GFP DNGW182 mutant at DIV7. The images are derived from the threshold MAP2 intensity of transfected neurons. Scale bar represents 50 microns. G) Intersection profile / Sholl curve of DIV11 cultured hippocampal neurons transfected with either control GFP or GFP DNGW182 mutant. Data: mean +/- SEM, n=22-25 neurons from 4 independent experiments. H) Quantification of the total dendritic length of DIV11 cultured hippocampal neurons transfected with either Control GFP or GFP DNGW182, n=22-23 neurons from 4 independent cultures, Unpaired t-test with Welch’s correction. I) Schematic showing the experimental procedure and timeline: Cultured hippocampal neurons were transfected on DIV3 followed by BDNF treatment for 48hrs starting from DIV5 onwards, and fixed on DIV7. J) Representative images of cultured hippocampal neurons transfected at DIV3 with either control GFP or GFP DNGW182. After transfection, the neurons were treated with BDNF (50ng/ml) on DIV 5 and were fixed on DIV7. Scale bar represents 50 microns. K) Sholl Quantification of the effects of GFP DNGW182 on BDNF-induced dendrite arborization formation of hippocampal neurons. Data: mean +/- SEM, n=25-30 neurons from 4 independent experiments, BDNF treatment resulted in increased dendritic intersections in GFP transfected neurons at 16-40 microns from the soma and resulted in increased intersections in GFP DNGW182 transfected neurons as well at 16-32 microns from the soma, Two way ANOVA followed by Bonferroni’s multiple comparisons test. L) Quantification of the total dendritic length of neurons described in Figure 3E , n=25-30 neurons from 3 independent experiments, One Way Anova followed by Bonferroni’s multiple comparisons test.
    Figure Legend Snippet: Overexpression of DNGW182 mutant decreases dendritic arborization during a distinct neurodevelopmental window. A) Schematic showing the experimental procedure and timeline: Cultured hippocampal neurons were transfected with either control GFP or GFP DNGW182 on DIV3 and fixed on DIV7, followed by immunostaining. Dendrites were identified with MAP2 staining. B) Representative micrographs of DIV7 cultured hippocampal neurons transfected with either control GFP or GFP DNGW182. The images are derived from the threshold MAP2 intensity of transfected neurons. Scale bar represents 50 microns. C) Intersection profile / Sholl curve of DIV7 cultured hippocampal neurons transfected with either control GFP or GFP DNGW182. Data: mean +/- SEM, n=32 neurons from 4 independent experiments, GFP DNGW182 overexpressing neurons had significantly more dendrites from GFP overexpressing neurons at 16-48 microns from the soma, Two way ANOVA followed by Bonferroni’s multiple comparisons test. D) Quantification of the total dendritic length of cultured hippocampal neurons transfected with either control GFP vector or DN GW182, n=27-32 neurons from 4 independent cultures, Mann-Whitney test. E) Schematic showing the experimental procedure and timeline: Cultured hippocampal neurons were transfected with either control GFP or GFP DNGW182 on DIV7 and fixed on DIV11, followed by immunostaining. Dendrites were identified with MAP2 staining F) Representative micrographs of DIV11 cultured hippocampal neurons transfected with either control GFP vector or GFP DNGW182 mutant at DIV7. The images are derived from the threshold MAP2 intensity of transfected neurons. Scale bar represents 50 microns. G) Intersection profile / Sholl curve of DIV11 cultured hippocampal neurons transfected with either control GFP or GFP DNGW182 mutant. Data: mean +/- SEM, n=22-25 neurons from 4 independent experiments. H) Quantification of the total dendritic length of DIV11 cultured hippocampal neurons transfected with either Control GFP or GFP DNGW182, n=22-23 neurons from 4 independent cultures, Unpaired t-test with Welch’s correction. I) Schematic showing the experimental procedure and timeline: Cultured hippocampal neurons were transfected on DIV3 followed by BDNF treatment for 48hrs starting from DIV5 onwards, and fixed on DIV7. J) Representative images of cultured hippocampal neurons transfected at DIV3 with either control GFP or GFP DNGW182. After transfection, the neurons were treated with BDNF (50ng/ml) on DIV 5 and were fixed on DIV7. Scale bar represents 50 microns. K) Sholl Quantification of the effects of GFP DNGW182 on BDNF-induced dendrite arborization formation of hippocampal neurons. Data: mean +/- SEM, n=25-30 neurons from 4 independent experiments, BDNF treatment resulted in increased dendritic intersections in GFP transfected neurons at 16-40 microns from the soma and resulted in increased intersections in GFP DNGW182 transfected neurons as well at 16-32 microns from the soma, Two way ANOVA followed by Bonferroni’s multiple comparisons test. L) Quantification of the total dendritic length of neurons described in Figure 3E , n=25-30 neurons from 3 independent experiments, One Way Anova followed by Bonferroni’s multiple comparisons test.

    Techniques Used: Over Expression, Mutagenesis, Cell Culture, Transfection, Immunostaining, Staining, Derivative Assay, Plasmid Preparation, MANN-WHITNEY

    9) Product Images from "A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators"

    Article Title: A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22031417

    Evaluation of the biosensor cells. Forskolin (10 µM) was used as a positive control and DMSO (1%) as a negative control. Means ± SEM of three individual experiments performed in duplicates are given. ( a ) Biosensor cells produced luminescence only in response to forskolin (10 µM). Luminescence response to CGS-21680 (1 µM) was not different from control (DMSO). ( b ) Cx43 transfection of biosensor (HeLa-GSL) cells did not affect luminescence responses. Statistical significance calculated with repeated measures 2-way ANOVA and Dunnet’s multiple comparisons test comparing treatments to control (DMSO, 1%). ** p
    Figure Legend Snippet: Evaluation of the biosensor cells. Forskolin (10 µM) was used as a positive control and DMSO (1%) as a negative control. Means ± SEM of three individual experiments performed in duplicates are given. ( a ) Biosensor cells produced luminescence only in response to forskolin (10 µM). Luminescence response to CGS-21680 (1 µM) was not different from control (DMSO). ( b ) Cx43 transfection of biosensor (HeLa-GSL) cells did not affect luminescence responses. Statistical significance calculated with repeated measures 2-way ANOVA and Dunnet’s multiple comparisons test comparing treatments to control (DMSO, 1%). ** p

    Techniques Used: Positive Control, Negative Control, Produced, Transfection

    10) Product Images from "Role of microRNA-130a in the pathogeneses of obstructive sleep apnea hypopnea syndrome-associated pulmonary hypertension by targeting the GAX gene"

    Article Title: Role of microRNA-130a in the pathogeneses of obstructive sleep apnea hypopnea syndrome-associated pulmonary hypertension by targeting the GAX gene

    Journal: Medicine

    doi: 10.1097/MD.0000000000006746

    Cell apoptosis of HUVECs in 5 groups after transfection using flow cytometry. Note: # = P
    Figure Legend Snippet: Cell apoptosis of HUVECs in 5 groups after transfection using flow cytometry. Note: # = P

    Techniques Used: Transfection, Flow Cytometry, Cytometry

    The tube formation of HUVECs in 5 groups after transfection under the phase contrast microscope (400×). Note: ∗ = P
    Figure Legend Snippet: The tube formation of HUVECs in 5 groups after transfection under the phase contrast microscope (400×). Note: ∗ = P

    Techniques Used: Transfection, Microscopy

    Related Articles

    Transfection:

    Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)
    Article Snippet: .. Cell transfections were performed using Lipofectamine 2000 (Invitrogen). .. LMB was purchased from LC Laboratories (MA, 01801).

    Article Title: PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3? and is required for metaphase-anaphase transition
    Article Snippet: Human Plk1 (including its mutants) and the above Plk1 orthologues were inserted into pCMV-Tag3B (Stratagene, La Jolla, CA). .. Plasmid transfection was performed with Lipofectamine LTX and PLUS Reagents according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). .. Protein purification We generated the recombinant baculovirus encoding the CA or KD mutant of human Akt1 using a combination of the GATEWAY vector conversion system and the Bac-to-Bac baculovirus expression system (Invitrogen).

    Article Title: Naturally Occurring Core Protein Mutations Compensate for the Reduced Replication Fitness of a Lamivudine-Resistant HBV Isolate
    Article Snippet: Cell culture and transfectionHepG2 cells and Huh7 cells are maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (Corning) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin. .. Cell transfection was conducted with Lipofectamine 2000 (Invitrogen) by following the manufacturer’s instruction. .. HBV RNA and DNA analysis Total cellular RNA was extracted from transfected cells by using the TRIzol reagent (Invitrogen) according to the manufacturer’s specifications.

    Article Title: Cytoplasmic Forms of Human T-Cell Leukemia Virus Type 1 Tax Induce NF-?B Activation
    Article Snippet: Immobilon membrane slices containing the tryptic fragments were then directly used for amino acid sequence determination as described previously ( ). .. HeLa cells were transfected by using Lipofectamine (Gibco-BRL) according to the manufacturer’s instructions. .. At 24 h posttransfection, cells were seeded on eight-well chamber slides (Nunc) and incubated for an additional 24 h at 37°C.

    Article Title: NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability
    Article Snippet: BLAST sequence analyses of the human genome database were carried out to ensure that all used siRNAs would not target other gene transcripts. .. SiRNA forward transfections were performed using lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) on two consecutive days and according to the instructions of the manufacturer, combined siRNA and plasmid transfections were carried out using Lipofectamine2000 (Invitrogen, Carlsbad, CA). .. SiRNA/lipofectamine RNAiMAX complexes were formed at a 1:1 ratio, and the concentration of siRNAs in transfections was 40 nM.

    Article Title: miR-223 regulates cell growth and targets proto-oncogenes in mycosis fungoides/cutaneous T-cell lymphoma
    Article Snippet: Luciferase Vector Generation and Luciferase Assay A 60mer of the 3′-UTR of TOX containing the miR-223 seed sequence and a 60mer with a mutated (base substitutions) seed sequence designed to prevent miR-223 from binding was cloned into pMIR-REPORT (Invitrogen). .. NIH-3T3 cells were transfected with 100 ng of pMIR-REPORT wild-type TOX 3′-UTR or mutant TOX 3′-UTR and 150 nM of miR-223 mimic or control RNA using Lipofectamine 2000 (Invitrogen). .. The cells were also co-transfected with 100 ng of pMIR-REPORT vector expressing a β-galactosidase control for normalizing transfection efficiency.

    Article Title: miR-505 inhibits cell growth and EMT by targeting MAP3K3 through the AKT-NFκB pathway in NSCLC cells
    Article Snippet: The human lung cancer cell lines A549, H1066, H460, H522 and SPC-A1, and the human normal lung epithelial cell BEAS-2B were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), and cultured in RPMI-1640 or Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin-streptomycin. .. Cell transfection was performed by Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. .. A549 cells or H460 cells (4×105 ) were planted in 6-well plates overnight at 37°C to ensure that cell confluence could reach 60-80% at the time of transfection.

    Article Title: Human Heat Shock Protein 105/110 kDa (Hsp105/110) Regulates Biogenesis and Quality Control of Misfolded Cystic Fibrosis Transmembrane Conductance Regulator at Multiple Levels *
    Article Snippet: CF airway epithelial IB3-1 cells ( ) were maintained in LHC-8 medium without gentamicin (Invitrogen), 5% fetal bovine serum, and 100 units/ml each of penicillin and streptomycin. .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen) or Effectine (Qiagen, Valencia, CA). .. HEK cells were transfected with Hsp105 shRNA or control shRNA construct.

    Plasmid Preparation:

    Article Title: PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3? and is required for metaphase-anaphase transition
    Article Snippet: Human Plk1 (including its mutants) and the above Plk1 orthologues were inserted into pCMV-Tag3B (Stratagene, La Jolla, CA). .. Plasmid transfection was performed with Lipofectamine LTX and PLUS Reagents according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). .. Protein purification We generated the recombinant baculovirus encoding the CA or KD mutant of human Akt1 using a combination of the GATEWAY vector conversion system and the Bac-to-Bac baculovirus expression system (Invitrogen).

    Article Title: NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability
    Article Snippet: BLAST sequence analyses of the human genome database were carried out to ensure that all used siRNAs would not target other gene transcripts. .. SiRNA forward transfections were performed using lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) on two consecutive days and according to the instructions of the manufacturer, combined siRNA and plasmid transfections were carried out using Lipofectamine2000 (Invitrogen, Carlsbad, CA). .. SiRNA/lipofectamine RNAiMAX complexes were formed at a 1:1 ratio, and the concentration of siRNAs in transfections was 40 nM.

    Mutagenesis:

    Article Title: miR-223 regulates cell growth and targets proto-oncogenes in mycosis fungoides/cutaneous T-cell lymphoma
    Article Snippet: Luciferase Vector Generation and Luciferase Assay A 60mer of the 3′-UTR of TOX containing the miR-223 seed sequence and a 60mer with a mutated (base substitutions) seed sequence designed to prevent miR-223 from binding was cloned into pMIR-REPORT (Invitrogen). .. NIH-3T3 cells were transfected with 100 ng of pMIR-REPORT wild-type TOX 3′-UTR or mutant TOX 3′-UTR and 150 nM of miR-223 mimic or control RNA using Lipofectamine 2000 (Invitrogen). .. The cells were also co-transfected with 100 ng of pMIR-REPORT vector expressing a β-galactosidase control for normalizing transfection efficiency.

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    Thermo Fisher streptavidin agarose beads
    HKR3 competes with FBI-1 at the proximal region of ARF , including three FBI-1 binding sites, in vitro . A , diagram of the endogenous ARF promoter. Sp1 and FBI-1 binding sites are indicated. +1, Tsp, transcription start site. B , oligonucleotide pull-down assays. Whole cell lysates of the HEK293 cells transfected with pcDNA3.1-HKR3-His and/or pcDNA3-FLAG-FBI-1 were incubated with <t>streptavidin-agarose</t> beads conjugated to biotinylated oligonucleotide probes: FRE#1 (bp, −303∼−273), FRE#2 (bp, −130∼−104), FRE#3 (bp, −103∼−76), FRE#4 (bp, −53∼−26). Proteins bound to the probes were precipitated by centrifugation and analyzed by Western blotting using the antibodies indicated. C , oligonucleotide pull-down DNA binding assays of recombinant GST, GST-POZ HKR3 , GST-ZF HKR3 , to the FRE#1, 2, 3, and 4. Recombinant proteins were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide FRE probes. The precipitates were analyzed by Western blotting using an anti-GST antibody.
    Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher gene exp dyx1c1 ccpg1 hs00370049 m1
    Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of <t>DYX1C1</t> and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.
    Gene Exp Dyx1c1 Ccpg1 Hs00370049 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cfse
    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous <t>CFSE-labeled</t> <t>CD8</t> + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.
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    Image Search Results


    HKR3 competes with FBI-1 at the proximal region of ARF , including three FBI-1 binding sites, in vitro . A , diagram of the endogenous ARF promoter. Sp1 and FBI-1 binding sites are indicated. +1, Tsp, transcription start site. B , oligonucleotide pull-down assays. Whole cell lysates of the HEK293 cells transfected with pcDNA3.1-HKR3-His and/or pcDNA3-FLAG-FBI-1 were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide probes: FRE#1 (bp, −303∼−273), FRE#2 (bp, −130∼−104), FRE#3 (bp, −103∼−76), FRE#4 (bp, −53∼−26). Proteins bound to the probes were precipitated by centrifugation and analyzed by Western blotting using the antibodies indicated. C , oligonucleotide pull-down DNA binding assays of recombinant GST, GST-POZ HKR3 , GST-ZF HKR3 , to the FRE#1, 2, 3, and 4. Recombinant proteins were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide FRE probes. The precipitates were analyzed by Western blotting using an anti-GST antibody.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Krüppel-related 3 (HKR3) Is a Novel Transcription Activator of Alternate Reading Frame (ARF) Gene *

    doi: 10.1074/jbc.M113.526855

    Figure Lengend Snippet: HKR3 competes with FBI-1 at the proximal region of ARF , including three FBI-1 binding sites, in vitro . A , diagram of the endogenous ARF promoter. Sp1 and FBI-1 binding sites are indicated. +1, Tsp, transcription start site. B , oligonucleotide pull-down assays. Whole cell lysates of the HEK293 cells transfected with pcDNA3.1-HKR3-His and/or pcDNA3-FLAG-FBI-1 were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide probes: FRE#1 (bp, −303∼−273), FRE#2 (bp, −130∼−104), FRE#3 (bp, −103∼−76), FRE#4 (bp, −53∼−26). Proteins bound to the probes were precipitated by centrifugation and analyzed by Western blotting using the antibodies indicated. C , oligonucleotide pull-down DNA binding assays of recombinant GST, GST-POZ HKR3 , GST-ZF HKR3 , to the FRE#1, 2, 3, and 4. Recombinant proteins were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide FRE probes. The precipitates were analyzed by Western blotting using an anti-GST antibody.

    Article Snippet: Cellular extracts or recombinant protein (GST-fusion protein) were incubated with biotinylated double-stranded oligonucleotides for 12 h. The mixtures were incubated with streptavidin-agarose beads (Thermo Fisher Scientific, Waltham, MA) for 4 h, washed with HKMG buffer five times, and precipitated by centrifugation.

    Techniques: Binding Assay, In Vitro, Transfection, Incubation, Centrifugation, Western Blot, Recombinant

    Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Binding Assay, Incubation, Quantitative RT-PCR

    The promoters of DYX1C1 , DCDC2 , and KIAA0319 contain functional X-box promoter motifs. A ) Location of putative X-box promoter motifs upstream of DYX1C1 , DCDC2 , and KIAA0319 identified in the bioinformatics screen. X-box sequence logos on the basis of human, mouse, dog, cow, and cat sequence alignments are shown above 3 well-conserved motifs. B ) Luciferase expression assays: pGL3 basic vector containing promoter sequences of DYX1C1 , DCDC2 , and KIAA0319 (containing WT or mutated X-box motifs) were transfected into starved hTERT-RPE1 and SH-SY5Y cells under normal growth conditions. Luciferase expression was measured and data are shown as relative ratio to the WT construct. Data are displayed as means ± sem. * P

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: The promoters of DYX1C1 , DCDC2 , and KIAA0319 contain functional X-box promoter motifs. A ) Location of putative X-box promoter motifs upstream of DYX1C1 , DCDC2 , and KIAA0319 identified in the bioinformatics screen. X-box sequence logos on the basis of human, mouse, dog, cow, and cat sequence alignments are shown above 3 well-conserved motifs. B ) Luciferase expression assays: pGL3 basic vector containing promoter sequences of DYX1C1 , DCDC2 , and KIAA0319 (containing WT or mutated X-box motifs) were transfected into starved hTERT-RPE1 and SH-SY5Y cells under normal growth conditions. Luciferase expression was measured and data are shown as relative ratio to the WT construct. Data are displayed as means ± sem. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Functional Assay, Sequencing, Luciferase, Expressing, Plasmid Preparation, Transfection, Construct

    Induction of ciliogenesis in hTERT-RPE1 cells affects the expression levels of DCGs, RFX TF genes, and ciliogenesis/cell-cycle marker genes. hTERT-RPE1 cells were starved for 6, 12, and 24 h to induce ciliogenesis. Fold-change of difference for each time point compared with 0 h is shown for DYX1C1 ( A ), DCDC2 ( B ), KIAA0319 ( C ), RFX1 ( D ), RFX2 ( E ), RFX3 ( F ), TUBA1A (tubulin α1a) ( G ), IFT57 (intraflagellar transport 57) ( H ), and CDK1 (cyclin-dependent kinase 1) ( I ). qRT-PCR data are summarized by using the ΔΔ C t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem .

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Induction of ciliogenesis in hTERT-RPE1 cells affects the expression levels of DCGs, RFX TF genes, and ciliogenesis/cell-cycle marker genes. hTERT-RPE1 cells were starved for 6, 12, and 24 h to induce ciliogenesis. Fold-change of difference for each time point compared with 0 h is shown for DYX1C1 ( A ), DCDC2 ( B ), KIAA0319 ( C ), RFX1 ( D ), RFX2 ( E ), RFX3 ( F ), TUBA1A (tubulin α1a) ( G ), IFT57 (intraflagellar transport 57) ( H ), and CDK1 (cyclin-dependent kinase 1) ( I ). qRT-PCR data are summarized by using the ΔΔ C t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem .

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Marker, Quantitative RT-PCR

    Subcellular localization of endogenous DYX1C1 ( A ) and DCDC2 ( B ) in hTERT-RPE1 cells. hTERT-RPE1 cells were grown to 80% confluence, serum starved for 24 h to induce ciliogenesis, then fixed and stained for DYX1C1, DCDC2, acetylated α-tubulin (cilia marker), or γ-tubulin (centrosome marker). Nuclei are stained with DRAQ5. Scale bars, 20 µm, 2 µm (insets).

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Subcellular localization of endogenous DYX1C1 ( A ) and DCDC2 ( B ) in hTERT-RPE1 cells. hTERT-RPE1 cells were grown to 80% confluence, serum starved for 24 h to induce ciliogenesis, then fixed and stained for DYX1C1, DCDC2, acetylated α-tubulin (cilia marker), or γ-tubulin (centrosome marker). Nuclei are stained with DRAQ5. Scale bars, 20 µm, 2 µm (insets).

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Staining, Marker

    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Incubation, Negative Control, Cell Culture, Labeling, Fluorescence, Transfection, Isolation, Plasmid Preparation, Flow Cytometry, Cytometry, Expressing

    Antigen targeted to early endosomes via CD40 is cross presented by both DC subsets with similar efficacy. (A) Anti-CD40 antibody intracellular trafficking. Day 1 BDCA1 + DCs (top) or BDCA3 + DCs (bottom) were fed with Alexa Fluor 488–labeled anti-CD40 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes or early endosomes were stained using anti-Lamp1 or anti-EEA1 (red), respectively. Plasma membrane was stained using anti–HLA-DR (blue) antibodies. Cells were then analyzed using confocal microscopy. Bars, 5 µm. Shown is one representative experiment of three. (B) Accumulation of anti-CD40 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-CD40 antibody continuously for 4–6 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 3 independent experiments). (C) Antigen cross presentation via CD40 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD40, or control isotype antibody conjugated to Flu-M1 (aa 55–72) at 1 µg/ml for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of more than five. (D) As in C; anti-CD40 and control isotype antibodies were conjugated to CMV-pp65 (aa 488–508) at the indicated doses. Shown is one representative experiment of two.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigen targeted to early endosomes via CD40 is cross presented by both DC subsets with similar efficacy. (A) Anti-CD40 antibody intracellular trafficking. Day 1 BDCA1 + DCs (top) or BDCA3 + DCs (bottom) were fed with Alexa Fluor 488–labeled anti-CD40 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes or early endosomes were stained using anti-Lamp1 or anti-EEA1 (red), respectively. Plasma membrane was stained using anti–HLA-DR (blue) antibodies. Cells were then analyzed using confocal microscopy. Bars, 5 µm. Shown is one representative experiment of three. (B) Accumulation of anti-CD40 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-CD40 antibody continuously for 4–6 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 3 independent experiments). (C) Antigen cross presentation via CD40 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD40, or control isotype antibody conjugated to Flu-M1 (aa 55–72) at 1 µg/ml for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of more than five. (D) As in C; anti-CD40 and control isotype antibodies were conjugated to CMV-pp65 (aa 488–508) at the indicated doses. Shown is one representative experiment of two.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Isolation, Cell Culture

    BDCA3 + DC exhibit an enhanced ability to cross present antigen delivered to lysosomes. (A) Antigen cross presentation via DEC205 by BDCA3 + DCs and BDCA1 + DCs. Day 1 DCs from HLA-A*0201 donors were fed with anti-DEC205 or control isotype antibody conjugated to Flu-M1 (aa 55–72) at the indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of n > 6. (B) As in A; anti-DEC205 and the control isotype antibodies were conjugated to CMV-pp65 (aa 488–508). Shown is one representative experiment of two. (C) Accumulation of anti-DEC205 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 antibody continuously for 4–6 h at 4 or 37°C. Results were analyzed by flow cytometry. 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 7 independent experiments). (D) Internalization of anti-DEC205 antibody. Day 1 DCs were incubated with Alexa Fluor 488–labeled anti-DEC205 antibody for 30 min at 4°C. The cells were then washed and cultured at 37°C for the indicated times. At each time point, cells were labeled with an Alexa Fluor 647–labeled anti–human IgG antibody to label remaining surface bound antibody. Cells were then analyzed by flow cytometry. The total DEC205 is Alexa Fluor 488–labeled antibody, whereas the surface DEC205 is the Alexa Fluor 647–labeled anti–human antibody. Shown is one representative experiment of four. (E) Anti-DEC205 antibody trafficking. Day 1 DCs were fed Alexa Fluor 488–labeled anti-DEC205 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes and cell membrane were stained using anti-Lamp1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bars, 7.5 µm. Shown is one representative experiment of five. (F) Detection of the indicated lysosomal proteases by Western blot of day 1 BDCA1 + DCs, BDCA3 + DCs, and mo-DCs. DC subsets were lysed and analyzed by Western blot for lysosomal protease expression. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: BDCA3 + DC exhibit an enhanced ability to cross present antigen delivered to lysosomes. (A) Antigen cross presentation via DEC205 by BDCA3 + DCs and BDCA1 + DCs. Day 1 DCs from HLA-A*0201 donors were fed with anti-DEC205 or control isotype antibody conjugated to Flu-M1 (aa 55–72) at the indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of n > 6. (B) As in A; anti-DEC205 and the control isotype antibodies were conjugated to CMV-pp65 (aa 488–508). Shown is one representative experiment of two. (C) Accumulation of anti-DEC205 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 antibody continuously for 4–6 h at 4 or 37°C. Results were analyzed by flow cytometry. 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 7 independent experiments). (D) Internalization of anti-DEC205 antibody. Day 1 DCs were incubated with Alexa Fluor 488–labeled anti-DEC205 antibody for 30 min at 4°C. The cells were then washed and cultured at 37°C for the indicated times. At each time point, cells were labeled with an Alexa Fluor 647–labeled anti–human IgG antibody to label remaining surface bound antibody. Cells were then analyzed by flow cytometry. The total DEC205 is Alexa Fluor 488–labeled antibody, whereas the surface DEC205 is the Alexa Fluor 647–labeled anti–human antibody. Shown is one representative experiment of four. (E) Anti-DEC205 antibody trafficking. Day 1 DCs were fed Alexa Fluor 488–labeled anti-DEC205 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes and cell membrane were stained using anti-Lamp1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bars, 7.5 µm. Shown is one representative experiment of five. (F) Detection of the indicated lysosomal proteases by Western blot of day 1 BDCA1 + DCs, BDCA3 + DCs, and mo-DCs. DC subsets were lysed and analyzed by Western blot for lysosomal protease expression. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Cell Culture, Labeling, Flow Cytometry, Cytometry, Incubation, Staining, Confocal Microscopy, Western Blot, Expressing

    Antigens escaping from lysosomes are cross presented equally by both DC subsets. (A) Number of IAV particles associated with DCs. Day 1 DCs were fed either with fusion-competent or -incompetent replication-defective (heat inactivated, HI) IAV for 6 h at 37°C. The cells were then washed and allowed to adhere to coverslips. After fixation and permeabilization, the cells were labeled with an anti-NP antibody. Cells were analyzed by confocal microscopy. Numbers of cells associated with IAV particles were counted and quantified. Shown is one representative experiment of three. (B) Cross presentation of fusion-competent and fusion-incompetent HI IAV. Day 1 DCs from HLA-A*0201 donors were fed with fusion-competent pH 7.4–treated HI IAV or fusion-incompetent pH 4.5–treated HI IAV for 6 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three. (C) Cross presentation of escape-competent and escape-incompetent KBMA L. monocytogenes . Day 1 DCs from HLA-A*0201 donors were fed for 1 h with escape-incompetent (LLO − ) and escape-competent (LLO + ) KBMA L. monocytogenes strains engineered to secrete ActAN100-Flu-M1 (aa 58–66) fusion protein. The cells were then washed and, as in B, cultured with autologous T cells to measure antigen cross presentation. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigens escaping from lysosomes are cross presented equally by both DC subsets. (A) Number of IAV particles associated with DCs. Day 1 DCs were fed either with fusion-competent or -incompetent replication-defective (heat inactivated, HI) IAV for 6 h at 37°C. The cells were then washed and allowed to adhere to coverslips. After fixation and permeabilization, the cells were labeled with an anti-NP antibody. Cells were analyzed by confocal microscopy. Numbers of cells associated with IAV particles were counted and quantified. Shown is one representative experiment of three. (B) Cross presentation of fusion-competent and fusion-incompetent HI IAV. Day 1 DCs from HLA-A*0201 donors were fed with fusion-competent pH 7.4–treated HI IAV or fusion-incompetent pH 4.5–treated HI IAV for 6 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three. (C) Cross presentation of escape-competent and escape-incompetent KBMA L. monocytogenes . Day 1 DCs from HLA-A*0201 donors were fed for 1 h with escape-incompetent (LLO − ) and escape-competent (LLO + ) KBMA L. monocytogenes strains engineered to secrete ActAN100-Flu-M1 (aa 58–66) fusion protein. The cells were then washed and, as in B, cultured with autologous T cells to measure antigen cross presentation. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Confocal Microscopy, Cell Culture

    Antigen delivered to early endosomes via CD40 is more efficiently cross presented by all DC subsets than antigen delivered to lysosomes via DEC205. (A) Antibody accumulation. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 or anti-CD40 antibody continuously for 3–4 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and resultant MFI was normalized to BDCA1 + DCs fed with anti-DEC205. MFI was also normalized for the number of fluorophores per antibody. Data shown are the mean MFI ± SD ( n = 5 independent experiments). (B and C) Antigen cross presentation via CD40 and DEC205 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-DEC205, anti-CD40, or control isotype antibodies conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cells were detected by staining with Flu-M1 (aa 58–66) pentamer. T cell proliferation was measured by CFSE dilution. The graphs shown in B show frequency of CFSE low , Flu-M1 (aa 58–66) pentamer-positive CD8 + T cells. In C, FACS plots of CD8 + T cells showing CFSE dilution in response to antigen presentation and numbers indicate frequency of CD8 + T cells in each quadrant. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigen delivered to early endosomes via CD40 is more efficiently cross presented by all DC subsets than antigen delivered to lysosomes via DEC205. (A) Antibody accumulation. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 or anti-CD40 antibody continuously for 3–4 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and resultant MFI was normalized to BDCA1 + DCs fed with anti-DEC205. MFI was also normalized for the number of fluorophores per antibody. Data shown are the mean MFI ± SD ( n = 5 independent experiments). (B and C) Antigen cross presentation via CD40 and DEC205 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-DEC205, anti-CD40, or control isotype antibodies conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cells were detected by staining with Flu-M1 (aa 58–66) pentamer. T cell proliferation was measured by CFSE dilution. The graphs shown in B show frequency of CFSE low , Flu-M1 (aa 58–66) pentamer-positive CD8 + T cells. In C, FACS plots of CD8 + T cells showing CFSE dilution in response to antigen presentation and numbers indicate frequency of CD8 + T cells in each quadrant. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Flow Cytometry, Cytometry, Isolation, Cell Culture, Staining, FACS

    Antigen targeted to early endosomes via CD11c is cross presented by both DC subsets with similar efficacy. (A) Anti-CD11c antibody intracellular trafficking. Day 1 BDCA1 + DCs were fed with Alexa Fluor 488–labeled anti-CD11c antibody (green) continuously for 3 h, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the early endosomes and plasma membrane were stained using EEA1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bar, 5 µm. Shown is one representative experiment of three. (B) Antigen cross presentation via CD11c in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD11c or control isotype antibody conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigen targeted to early endosomes via CD11c is cross presented by both DC subsets with similar efficacy. (A) Anti-CD11c antibody intracellular trafficking. Day 1 BDCA1 + DCs were fed with Alexa Fluor 488–labeled anti-CD11c antibody (green) continuously for 3 h, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the early endosomes and plasma membrane were stained using EEA1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bar, 5 µm. Shown is one representative experiment of three. (B) Antigen cross presentation via CD11c in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD11c or control isotype antibody conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Staining, Confocal Microscopy, Isolation, Cell Culture