primary human dermal fibroblasts  (ATCC)


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    ATCC primary human dermal fibroblasts
    Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from <t>fibroblasts</t> (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.
    Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis"

    Article Title: Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms25094939

    Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.
    Figure Legend Snippet: Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.

    Techniques Used: Expressing, Western Blot

    cytotoxicity assays primary adult human dermal fibroblasts  (ATCC)


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    ATCC cytotoxicity assays primary adult human dermal fibroblasts
    Cytotoxicity Assays Primary Adult Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    primary adult human dermal fibroblasts  (ATCC)


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    ATCC primary adult human dermal fibroblasts
    Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal <t>fibroblasts;</t> CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.
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    1) Product Images from "An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles"

    Article Title: An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-59866-y

    Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal fibroblasts; CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.
    Figure Legend Snippet: Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal fibroblasts; CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.

    Techniques Used: MTT Assay, Incubation, Concentration Assay, Saline

    primary normal human dermal fibroblast fibh cells  (ATCC)


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    ATCC primary normal human dermal fibroblast fibh cells
    Primary Normal Human Dermal Fibroblast Fibh Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    adult primary human dermal fibroblasts  (ATCC)


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    ATCC adult primary human dermal fibroblasts
    Adult Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    primary human dermal fibroblasts hdf  (ATCC)


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    ATCC primary human dermal fibroblasts hdf
    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal <t>fibroblasts</t> <t>(HDF)</t> were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.
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    1) Product Images from "Chloride Homeostasis Regulates cGAS-STING Signaling"

    Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

    Journal: bioRxiv

    doi: 10.1101/2024.04.08.588475

    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.
    Figure Legend Snippet: ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

    Techniques Used: Activation Assay, Cell Culture, Activity Assay, Transfection, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Staining

    primary human dermal fibroblasts  (ATCC)


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    ATCC primary human dermal fibroblasts
    In vitro biocompatibility testing of decellularized flaps using <t>fibroblasts.</t> ( A ) H&E stain showing cell attachment and penetration through the dermal side of the scaffold on days 1, 4, and 7 after cell seeding. Arrows indicate cells found deep in the tissue. ( B ) Cell proliferation as measured with Presto Blue assay. ( C ) The quantification of cell penetration depth as distance from the epidermal surface of the scaffold over 7 days of culture. * p < 0.05, **** p < 0.0001 via Student’s t -test, n = 3. (Scale bars, main figure = 200 μm; scale bars, zoomed in image = 50 μm).
    Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Optimized Decellularization of a Porcine Fasciocutaneaous Flap"

    Article Title: Optimized Decellularization of a Porcine Fasciocutaneaous Flap

    Journal: Bioengineering

    doi: 10.3390/bioengineering11040321

    In vitro biocompatibility testing of decellularized flaps using fibroblasts. ( A ) H&E stain showing cell attachment and penetration through the dermal side of the scaffold on days 1, 4, and 7 after cell seeding. Arrows indicate cells found deep in the tissue. ( B ) Cell proliferation as measured with Presto Blue assay. ( C ) The quantification of cell penetration depth as distance from the epidermal surface of the scaffold over 7 days of culture. * p < 0.05, **** p < 0.0001 via Student’s t -test, n = 3. (Scale bars, main figure = 200 μm; scale bars, zoomed in image = 50 μm).
    Figure Legend Snippet: In vitro biocompatibility testing of decellularized flaps using fibroblasts. ( A ) H&E stain showing cell attachment and penetration through the dermal side of the scaffold on days 1, 4, and 7 after cell seeding. Arrows indicate cells found deep in the tissue. ( B ) Cell proliferation as measured with Presto Blue assay. ( C ) The quantification of cell penetration depth as distance from the epidermal surface of the scaffold over 7 days of culture. * p < 0.05, **** p < 0.0001 via Student’s t -test, n = 3. (Scale bars, main figure = 200 μm; scale bars, zoomed in image = 50 μm).

    Techniques Used: In Vitro, Staining, Cell Attachment Assay

    primary human dermal fibroblasts  (ATCC)


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    ATCC primary human dermal fibroblasts
    Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    normal primary human dermal fibroblasts  (ATCC)


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    ATCC normal primary human dermal fibroblasts
    αSMA gene expression and protein levels are reduced in TGFβ1-activated <t>fibroblasts</t> following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Normal Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ketotifen directly modifies the fibrotic response of human skin fibroblasts"

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-57776-7

    αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Figure Legend Snippet: αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

    Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.
    Figure Legend Snippet: Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.

    Techniques Used: Cell Culture, Immunofluorescence, Staining

    Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.
    Figure Legend Snippet: Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Techniques Used: Expressing

    Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.
    Figure Legend Snippet: Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Techniques Used: Expressing, Quantitative RT-PCR, Residue, Western Blot, Binding Assay

    Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.
    Figure Legend Snippet: Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.

    Techniques Used: In Vitro, Expressing

    Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.
    Figure Legend Snippet: Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Techniques Used: Residue, Binding Assay

    primary human dermal fibroblasts  (ATCC)


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    Structured Review

    ATCC primary human dermal fibroblasts
    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal <t>fibroblasts.</t> At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
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    Images

    1) Product Images from "Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors"

    Article Title: Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-54941-w

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Figure Legend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Figure Legend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.
    Figure Legend Snippet: HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

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    ATCC primary human dermal fibroblasts
    Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from <t>fibroblasts</t> (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.
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    ATCC cytotoxicity assays primary adult human dermal fibroblasts
    Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from <t>fibroblasts</t> (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.
    Cytotoxicity Assays Primary Adult Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC primary adult human dermal fibroblasts
    Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal <t>fibroblasts;</t> CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.
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    ATCC primary normal human dermal fibroblast fibh cells
    Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal <t>fibroblasts;</t> CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.
    Primary Normal Human Dermal Fibroblast Fibh Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC adult primary human dermal fibroblasts
    Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal <t>fibroblasts;</t> CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.
    Adult Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC primary human dermal fibroblasts hdf
    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal <t>fibroblasts</t> <t>(HDF)</t> were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.
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    ATCC normal primary human dermal fibroblasts
    αSMA gene expression and protein levels are reduced in TGFβ1-activated <t>fibroblasts</t> following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.
    Normal Primary Human Dermal Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.

    Journal: International Journal of Molecular Sciences

    Article Title: Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis

    doi: 10.3390/ijms25094939

    Figure Lengend Snippet: Expression of α-ENaC as a 75 kDa protein. ( A ) Western blot assays were performed on total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, platelets (Pts), and neutrophils (Nphils) using an anti-α-ENaC polyclonal antibody, revealing a corresponding band at ~75 kDa (arrow). ( B ) Bands at ~75 kDa were detected in total lysates from fibroblasts (Fbs), Human Embryonic Kidney (HEK-293) cells, C2C12 cells, platelets (Pts), and neutrophils (Nphils). No bands were observed in the protein extracts of Escherichia coli C41. No bands were observed with the exposure to the anti-IgG HRP secondary antibody in the absence of primary antibody.

    Article Snippet: Primary human dermal fibroblasts (AG08469 Coriell), HEK293 cells (CRL1573 ATCC), and C2C12 (CRL1772 ATCC) were cultured in MEM, DMEM/F-12, and DMEM, respectively (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Western Blot

    Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal fibroblasts; CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.

    Journal: Scientific Reports

    Article Title: An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles

    doi: 10.1038/s41598-024-59866-y

    Figure Lengend Snippet: Cytotoxicities of T7 phages and AgNPs to aHDFs and Jurkat E6-1 cells, as determined by MTT assay. Panels show the cytotoxicities of T7wt and T7Ag-XII phages, AgNPs, and T7Ag-XII phages in combination with AgNPs. ( A , C , E ): aHDFs after 24, 48, and 72 h of incubation, respectively; ( B , D , F ): Jurkat E6-1 cells after 24, 48, and 72 h of incubation, respectively. The working concentration of T7wt or T7Ag-XII phages was kept at 1 × 10 9 pfu/mL. The working concentration of AgNPs: AgNPs −1 was 0.1 mg/mL; the variations in AgNPs working concentrations decreased by an order of magnitude indicated as AgNPs −2 , AgNPs −3 . The working concentration of the combination of T7-XII and AgNPs: T7Ag-XII AgNPs −1 was 0.1 mg/mL for AgNPs and 1 × 10 9 pfu/mL for T7Ag-XII phages; in the variations in the combination of phages and AgNPs, working concentrations of AgNPs decreased by an order of magnitude indicated as T7Ag-XII AgNPs −1 , T7Ag-XII AgNPs −2 , T7Ag-XII AgNPs −3 , and the concentration of phages was constant (1 × 10 9 pfu/mL). Data are shown as mean values of three biological replicates, and standard deviations are represented by error bars. Statistical analysis (one-way ANOVA) was carried out and significant P -values are presented. AgNPs , Silver nanoparticles; aHDFs , Adult human dermal fibroblasts; CTRL , Cells treated with fresh medium; CTRL TBS , Cells treated with Tris-buffered saline; CTRL Triton X , Cells treated with Triton X; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T7Ag-XII , T7 phages displaying the RFEHPAVPRTEM peptide; T7wt , Wild-type T7 phages.

    Article Snippet: Primary adult human dermal fibroblasts (American Type Culture Collection [ATCC]: PCS-201–012™) and human Jurkat T lymphoblasts, clone E6-1 (ATCC: TIB-152™) were used for cytotoxicity assays.

    Techniques: MTT Assay, Incubation, Concentration Assay, Saline

    ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

    Journal: bioRxiv

    Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

    doi: 10.1101/2024.04.08.588475

    Figure Lengend Snippet: ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

    Article Snippet: Primary Human Dermal Fibroblasts (HDF) were purchased from ATCC (Manassas, VA, USA).

    Techniques: Activation Assay, Cell Culture, Activity Assay, Transfection, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Staining

    αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. ( A , B ) HDFa and ( C , D ) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA ( ACTA2 ) was measured using RT-qPCR and normalized to housekeeping gene HPRT . HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. ( E ) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. . ( F ) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 ( G ) and fibronectin ( H ) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

    Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Ketotifen treatment reduced αSMA fibres in TGFβ1-activated fibroblasts. HDFa cells were cultured on poly- d -lysine-coated coverslips and treated for 48 h with media, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A ) Representative images of immunofluorescence staining (200X total magnification) probing for αSMA and DAPI are shown on the different groups of HDFa cells. ( B ) Quantification of αSMA+ staining (green) was plotted per DAPI+ cell (blue). Measurements for each biological replicate were averaged from five microscopic fields of view. Data shown as mean ± SEM. n = 3 per treatment condition. ** p < 0.01; *** p < 0.001; **** p < 0.0001. αSMA, alpha-smooth muscle actin, DAPI, 4′, 6-diamidino-2-phenylindole, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta 1.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Cell Culture, Immunofluorescence, Staining

    Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Cytoskeletal-associated genes CNN1 and TAGLN are reduced with ketotifen treatment. HDFa and WS1 fibroblasts cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. CNN1 and TAGLN expression were assessed under 10 μM ketotifen (HDFa: A , B ; WS1: E , F ) and 25 μM ketotifen (HDFa: C , D ; WS1: G , H ) conditions. Data shown as mean ± SEM. n = 3–6 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CNN1 calponin 1, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAGLN transgelin, TGFβ1 transforming growth factor-beta 1.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing

    Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. ( A , B ) Gene expression of YAP ( YAP1 ) and TAZ ( WWTR1 ) were assessed using RT-qPCR and normalized to housekeeping gene HPRT . ( C ) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. ( D ) TAZ protein was measured and plotted in the same manner. ( E ) Representative images of the blots are shown. ( F ) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; ** p < 0.01; *** p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Expressing, Quantitative RT-PCR, Residue, Western Blot, Binding Assay

    Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Ketotifen fumarate treatment impaired TGFβ1-stimulated fibroblast contraction in an in vitro model of fibroblast-populated gel contraction. ( A ) Representative images of fibroblast-populated collagen gels in DF10 (DMEM, with 10% FBS) culture media with or without ketotifen fumarate at 0- and 4 h post-treatment. ( B ) Contracture of activated fibroblast-populated collagen gels was determined and plotted over the course of 24 h as mean gel area in normal or ketotifen-supplemented media. ( C ) TGFβ1 or TGFβ1 and ketotifen fumarate-treated fibroblasts were seeded into collagen gels and contracture measured in normal culture media. ( D ) ACTA2 is significantly reduced in ketotifen-treated TGFβ1-activated fibroblasts compared to TGFβ1 treatment at 4 h. Gene expression data is normalized to housekeeping gene HPRT . Data shown as mean ± SEM. n = 3 per treatment condition. * p < 0.05; *** p < 0.001; **** p < 0.0001. DF10 10% FBS-DMEM, DF10-K 10% FBS-DMEM with ketotifen fumarate, TGFβ1 transforming growth factor-beta 1, TK25 transforming growth factor-beta 1 with 25 μM ketotifen.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: In Vitro, Expressing

    Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Journal: Scientific Reports

    Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts

    doi: 10.1038/s41598-024-57776-7

    Figure Lengend Snippet: Schematic of the effects of ketotifen fumarate on fibroblast phenotype and associated cellular mechanisms. ( A ) Ketotifen fumarate inhibits the effects of TGFβ1 on fibroblasts, decreasing αSMA gene and protein levels, cytoskeletal-associated genes, and contractility. ( B ) Ketotifen fumarate treatment reduces ACTA2 , CNN1 , and TAGLN , and decreases TAZ gene and protein levels. Increased phosphorylation of YAP at serine residue 127 is also seen, promoting cytosolic localization. Ketotifen also affects the PI3K-AKT pathway by reducing phosphorylation of AKT at serine residue 473. AKT protein kinase B, αSMA alpha-smooth muscle actin, CNN1 calponin 1, PI3K phosphatidylinositol-3-kinase, TAGLN transgelin, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.

    Article Snippet: Normal primary human dermal fibroblasts (HDFa, PCS-201-012, ATCC, Manassas, VA; isolated from skin of a 27-year-old Asian male) and normal human WS1 dermal fibroblasts (CRL-1502, ATCC; isolated from skin of a Black female at 12-weeks of gestation) were used for in vitro experiments.

    Techniques: Residue, Binding Assay