dopaminergic human neuroblastoma cell line sh sy5y  (ATCC)


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    ATCC dopaminergic human neuroblastoma cell line sh sy5y
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Dopaminergic Human Neuroblastoma Cell Line Sh Sy5y, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells"

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232315255

    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Expressing, Incubation, Immunofluorescence, Staining, Western Blot

    APM protects MPP + -induced mitochondria-dependent neurotoxicity in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h or 24 h. Expression of cleaved-Caspase3 and cleaved-PARP in rat embryo primary mesencephalic neurons ( A ) and SH-SY5Y cells ( B ) were detected by immunoblotting. βActin was used to confirm equal sample loading. Arrows: cleaved form. ( C ) SH-SY5Y cells were evaluated by fluorescence microscopy on the basis of morphological criteria after TUNEL staining (upper, scale bars: 100 μm) and JC-1 mitochondrial staining (lower, scale bars: 50 μm) and immunofluorescence positive cells were quantified by densitometric analysis ( D ). Nuclei were stained with DAPI. APM regulates mitochondrial apoptotic proteins ( E ). VDAC was used as mitochondrial loading control. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM protects MPP + -induced mitochondria-dependent neurotoxicity in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h or 24 h. Expression of cleaved-Caspase3 and cleaved-PARP in rat embryo primary mesencephalic neurons ( A ) and SH-SY5Y cells ( B ) were detected by immunoblotting. βActin was used to confirm equal sample loading. Arrows: cleaved form. ( C ) SH-SY5Y cells were evaluated by fluorescence microscopy on the basis of morphological criteria after TUNEL staining (upper, scale bars: 100 μm) and JC-1 mitochondrial staining (lower, scale bars: 50 μm) and immunofluorescence positive cells were quantified by densitometric analysis ( D ). Nuclei were stained with DAPI. APM regulates mitochondrial apoptotic proteins ( E ). VDAC was used as mitochondrial loading control. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Expressing, Western Blot, Fluorescence, Microscopy, TUNEL Assay, Staining, Immunofluorescence

    APM inhibited MPP + -induced SK channels in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM or BAPTA-AM (5 μM) for 1 h and then treated with MPP + for 24 h. ( A ) Intracellular Ca 2+ overload positive cells of rat embryo primary mesencephalic neurons were evaluated using fluorescence microscopy using FLUOFORTE staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). APM significantly inhibit MPP + -induced Kca2.2 and pCaMKⅡ expression in rat embryo primary mesencephalic neurons ( B ) and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM inhibited MPP + -induced SK channels in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM or BAPTA-AM (5 μM) for 1 h and then treated with MPP + for 24 h. ( A ) Intracellular Ca 2+ overload positive cells of rat embryo primary mesencephalic neurons were evaluated using fluorescence microscopy using FLUOFORTE staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). APM significantly inhibit MPP + -induced Kca2.2 and pCaMKⅡ expression in rat embryo primary mesencephalic neurons ( B ) and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Fluorescence, Microscopy, Staining, Expressing, Western Blot, Immunofluorescence

    APM alleviated MPP + -induced neuroinflammatory response, oxidative stress, and ER stress. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. Expression of inflammatory cytokines ( A ), HIF1α, NOX2, GRP78, and CHOP ( B ) in SH-SY5Y cells were analyzed by immunoblotting. ( C ) Expression of TNFα, NOX2, and CHOP in rat embryo primary mesencephalic neurons were confirmed by immunoblotting. βActin was used to confirm equal sample loading. ( D ) ROS generation in rat embryo primary mesencephalic neurons was detected by DCF-DA staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM alleviated MPP + -induced neuroinflammatory response, oxidative stress, and ER stress. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. Expression of inflammatory cytokines ( A ), HIF1α, NOX2, GRP78, and CHOP ( B ) in SH-SY5Y cells were analyzed by immunoblotting. ( C ) Expression of TNFα, NOX2, and CHOP in rat embryo primary mesencephalic neurons were confirmed by immunoblotting. βActin was used to confirm equal sample loading. ( D ) ROS generation in rat embryo primary mesencephalic neurons was detected by DCF-DA staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Expressing, Western Blot, Staining, Immunofluorescence

    APM strongly inhibited MPP + -induced phosphorylation of MAPK-ERK/p65/STAT3 signaling pathway. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h. Phosphorylation of ERK in SH-SY5Y cells ( A ) and rat embryo primary mesencephalic neurons ( B ) were analyzed by immunoblotting. MPP + -induced NFκB-p65 and STAT3 translocation in SH-SY5Y cells ( C ) and rat embryo primary mesencephalic neurons ( D ) were analyzed by immunoblotting. βActin was used to confirm equal sample loading. ( E ) The DNA-binding activity of NFκB-p65 and STAT3 in nuclear extracts was measured using EMSA. ( F ) Immunofluorescence double staining for TH (green) and pp65 (red) localization, and TH (green) and pSTAT3 (red) localization, in rat embryo primary mesencephalic neurons. Scale bars: 50 μm. Nuclei were stained with DAPI (blue). Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM strongly inhibited MPP + -induced phosphorylation of MAPK-ERK/p65/STAT3 signaling pathway. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h. Phosphorylation of ERK in SH-SY5Y cells ( A ) and rat embryo primary mesencephalic neurons ( B ) were analyzed by immunoblotting. MPP + -induced NFκB-p65 and STAT3 translocation in SH-SY5Y cells ( C ) and rat embryo primary mesencephalic neurons ( D ) were analyzed by immunoblotting. βActin was used to confirm equal sample loading. ( E ) The DNA-binding activity of NFκB-p65 and STAT3 in nuclear extracts was measured using EMSA. ( F ) Immunofluorescence double staining for TH (green) and pp65 (red) localization, and TH (green) and pSTAT3 (red) localization, in rat embryo primary mesencephalic neurons. Scale bars: 50 μm. Nuclei were stained with DAPI (blue). Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Western Blot, Translocation Assay, Binding Assay, Activity Assay, Immunofluorescence, Double Staining, Staining

    human dopaminergic neuroblastoma cell line sh sh5y  (ATCC)


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    ATCC human dopaminergic neuroblastoma cell line sh sh5y
    Human Dopaminergic Neuroblastoma Cell Line Sh Sh5y, 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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    human dopaminergic neuroblastoma sh sy5y cell line  (ATCC)


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    ATCC human dopaminergic neuroblastoma sh sy5y cell line
    Human Dopaminergic Neuroblastoma Sh Sy5y Cell Line, 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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    human dopaminergic neuroblastoma sh sy5y cell lines  (ATCC)


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    ATCC human dopaminergic neuroblastoma sh sy5y cell lines
    Human Dopaminergic Neuroblastoma Sh Sy5y Cell Lines, 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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    human dopaminergic neuroblastoma sh sy5y cell lines  (ATCC)


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    ATCC human dopaminergic neuroblastoma sh sy5y cell lines
    The concentration-dependent effect of Aβ on inflammasome protein and proinflammatory cytokine expression in <t>SH-SY5Y</t> cells. Cells were incubated with various concentration (01, 1 and 2 µM) of Aβ for 24 min. Western blot analysis was used to determine the expression levels of ( a ) NLRP3, ( b ) ASC, ( c ) Caspase 1, ( d ) pro-IL-1β, ( e ) IL-1β, ( f ) pro-IL-18, ( g ) IL-18 and ( h ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3–4 (*, **, ***, ****denote statistical significance at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 compared to the control group, respectively).
    Human Dopaminergic Neuroblastoma Sh Sy5y Cell Lines, 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 "The role of melatonin in amyloid beta-induced inflammation mediated by inflammasome signaling in neuronal cell lines"

    Article Title: The role of melatonin in amyloid beta-induced inflammation mediated by inflammasome signaling in neuronal cell lines

    Journal: Scientific Reports

    doi: 10.1038/s41598-023-45220-1

    The concentration-dependent effect of Aβ on inflammasome protein and proinflammatory cytokine expression in SH-SY5Y cells. Cells were incubated with various concentration (01, 1 and 2 µM) of Aβ for 24 min. Western blot analysis was used to determine the expression levels of ( a ) NLRP3, ( b ) ASC, ( c ) Caspase 1, ( d ) pro-IL-1β, ( e ) IL-1β, ( f ) pro-IL-18, ( g ) IL-18 and ( h ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3–4 (*, **, ***, ****denote statistical significance at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 compared to the control group, respectively).
    Figure Legend Snippet: The concentration-dependent effect of Aβ on inflammasome protein and proinflammatory cytokine expression in SH-SY5Y cells. Cells were incubated with various concentration (01, 1 and 2 µM) of Aβ for 24 min. Western blot analysis was used to determine the expression levels of ( a ) NLRP3, ( b ) ASC, ( c ) Caspase 1, ( d ) pro-IL-1β, ( e ) IL-1β, ( f ) pro-IL-18, ( g ) IL-18 and ( h ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3–4 (*, **, ***, ****denote statistical significance at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 compared to the control group, respectively).

    Techniques Used: Concentration Assay, Expressing, Incubation, Western Blot

    The concentration-dependent effect of melatonin on Aβ-induced inflammasome proteins expression in SH-SY5Y cells. Cells were pretreated with 1 µM or 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of ( a ) inflammasome proteins including NLRP3, ( b ) ASC, ( c ) pro-Caspase 1 and ( d ) cleaved-caspase 1. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. one-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3–4 (*, **, ****denote statistical significance at p < 0.05, p < 0.01, and p < 0.0001 compared to the control group, and ## , ### , #### denote statistical significance at p < 0.01, p < 0.001, and p < 0.0001 compared to the Aβ treatment group, respectively).
    Figure Legend Snippet: The concentration-dependent effect of melatonin on Aβ-induced inflammasome proteins expression in SH-SY5Y cells. Cells were pretreated with 1 µM or 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of ( a ) inflammasome proteins including NLRP3, ( b ) ASC, ( c ) pro-Caspase 1 and ( d ) cleaved-caspase 1. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. one-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3–4 (*, **, ****denote statistical significance at p < 0.05, p < 0.01, and p < 0.0001 compared to the control group, and ## , ### , #### denote statistical significance at p < 0.01, p < 0.001, and p < 0.0001 compared to the Aβ treatment group, respectively).

    Techniques Used: Concentration Assay, Expressing, Western Blot

    A representative image showing NLRP3 immunostaining in Aβ42 and melatonin-treated SH-SY5Y cell cultures. Control cells were incubated in serum free media for 24 h. Cells were treated with 1 µM melatonin for 24 h. Cells were treated with 1 µM Aβ42 for 24 h, and cells were pretreated with 1 µM melatonin for 2 h prior to 24 h of 1 µM Aβ42 treatment. The red color indicates NLRP3-positive immunostaining using Alexa 488-conjugated goat anti-rabbit IgG and DAPI nuclear staining (1:2000, blue color) on cover slips. The scale bar equals 30 μm.
    Figure Legend Snippet: A representative image showing NLRP3 immunostaining in Aβ42 and melatonin-treated SH-SY5Y cell cultures. Control cells were incubated in serum free media for 24 h. Cells were treated with 1 µM melatonin for 24 h. Cells were treated with 1 µM Aβ42 for 24 h, and cells were pretreated with 1 µM melatonin for 2 h prior to 24 h of 1 µM Aβ42 treatment. The red color indicates NLRP3-positive immunostaining using Alexa 488-conjugated goat anti-rabbit IgG and DAPI nuclear staining (1:2000, blue color) on cover slips. The scale bar equals 30 μm.

    Techniques Used: Immunostaining, Incubation, Staining

    The concentration-dependent effect of melatonin on Aβ-induced cytokine expression in SH-SY5Y cells. Cells were pretreated with 1 µM or 10 µM melatonin for 2 h and followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of cytokines including ( a ) pro-IL-1β, ( b ) IL-1β, ( c ) pro-IL-18, ( d ) IL-18 and ( e ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3 (*, ****denote statistical significance at p < 0.05 and 0.0001 compared to the control group, and # , ## denote statistical significance at p < 0.05 and p < 0.01, compared to the Aβ treatment group, respectively).
    Figure Legend Snippet: The concentration-dependent effect of melatonin on Aβ-induced cytokine expression in SH-SY5Y cells. Cells were pretreated with 1 µM or 10 µM melatonin for 2 h and followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of cytokines including ( a ) pro-IL-1β, ( b ) IL-1β, ( c ) pro-IL-18, ( d ) IL-18 and ( e ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 3 (*, ****denote statistical significance at p < 0.05 and 0.0001 compared to the control group, and # , ## denote statistical significance at p < 0.05 and p < 0.01, compared to the Aβ treatment group, respectively).

    Techniques Used: Concentration Assay, Expressing, Western Blot

    The effect of melatonin on Aβ-induced cytokine levels in SH-SY5Y cells. Cell were pretreated with 1 µM or 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Alpha-LISA analysis was used to determine the expression levels of cytokines including ( a ) IL-1β, ( b ) IL-18 and ( c ) TNF-α. Cell solutions were examined by using an Alpha-capable instrument. The values were calculated as the pg/µL of the standard value. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 4 (**, ***denote statistical significance at p < 0.01 and p < 0.001 compared to the control group, respectively, and # denotes statistical significance at p < 0.05 compared to the Aβ treatment group).
    Figure Legend Snippet: The effect of melatonin on Aβ-induced cytokine levels in SH-SY5Y cells. Cell were pretreated with 1 µM or 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Alpha-LISA analysis was used to determine the expression levels of cytokines including ( a ) IL-1β, ( b ) IL-18 and ( c ) TNF-α. Cell solutions were examined by using an Alpha-capable instrument. The values were calculated as the pg/µL of the standard value. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 4 (**, ***denote statistical significance at p < 0.01 and p < 0.001 compared to the control group, respectively, and # denotes statistical significance at p < 0.05 compared to the Aβ treatment group).

    Techniques Used: Expressing

    Protective effects of melatonin against Aβ-induced cytokines via the melatonin receptor in SH-SY5Y cells. Cells were treated with 1 µM luzindole 30 min prior to pretreatment with 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of inflammatory cytokines including ( a ) pro-IL-1β, ( b ) IL-1β, ( c ) pro-IL-18, ( d ) IL-18 and ( e ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 4 (*, ****denote statistical significance at p < 0.05, p < 0.0001 compared to the control group; ## , ### denote statistical significance at p < 0.01 and p < 0.001 compared to the Aβ treatment group, respectively; and ƒ and ff denote statistical significance at p < 0.05, 0.01 compared to melatonin pretreatment group).
    Figure Legend Snippet: Protective effects of melatonin against Aβ-induced cytokines via the melatonin receptor in SH-SY5Y cells. Cells were treated with 1 µM luzindole 30 min prior to pretreatment with 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of inflammatory cytokines including ( a ) pro-IL-1β, ( b ) IL-1β, ( c ) pro-IL-18, ( d ) IL-18 and ( e ) TNF-α. The band densities were normalized to actin. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 4 (*, ****denote statistical significance at p < 0.05, p < 0.0001 compared to the control group; ## , ### denote statistical significance at p < 0.01 and p < 0.001 compared to the Aβ treatment group, respectively; and ƒ and ff denote statistical significance at p < 0.05, 0.01 compared to melatonin pretreatment group).

    Techniques Used: Western Blot, Expressing

    Effects of cytokines release via the inflammasome in Aβ-treated SH-SY5Y cell. Cell were treated with 1 µM INF-4E (NLRP3, caspase 1 inhibitor) 30 min prior to pretreatment with 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of ( a ) pro-IL-1β, ( b ) cleaved-IL-1β, ( c ) cleaved/pro-IL-1β, ( d ) pro-IL-18, ( e ) cleaved-IL-18, ( f ) cleaved/pro-IL-18 and ( g ) TNF-α and ( h ) Gasdermin D. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 4 (*, **, ****denote statistical significance at p < 0.05, p < 0.01, and p < 0.0001 compared to the control group and # , ## , ### denote statistical significance at p < 0.05, p < 0.01 and p < 0.001 compared to the Aβ treatment group, respectively).
    Figure Legend Snippet: Effects of cytokines release via the inflammasome in Aβ-treated SH-SY5Y cell. Cell were treated with 1 µM INF-4E (NLRP3, caspase 1 inhibitor) 30 min prior to pretreatment with 10 µM melatonin for 2 h followed by 1 µM Aβ treatment. Western blot analysis was used to determine the expression levels of ( a ) pro-IL-1β, ( b ) cleaved-IL-1β, ( c ) cleaved/pro-IL-1β, ( d ) pro-IL-18, ( e ) cleaved-IL-18, ( f ) cleaved/pro-IL-18 and ( g ) TNF-α and ( h ) Gasdermin D. The ratios were calculated as a percentage of the respective value in the control group. The data are expressed as the means ± S.E.M. One-way ANOVA and Tukey's pos-hoc test were performed for statistical analysis. N = 4 (*, **, ****denote statistical significance at p < 0.05, p < 0.01, and p < 0.0001 compared to the control group and # , ## , ### denote statistical significance at p < 0.05, p < 0.01 and p < 0.001 compared to the Aβ treatment group, respectively).

    Techniques Used: Western Blot, Expressing

    human dopaminergic neuroblastoma cell line sh sh5y  (ATCC)


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    ATCC human dopaminergic neuroblastoma cell line sh sh5y
    Human Dopaminergic Neuroblastoma Cell Line Sh Sh5y, 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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    human dopaminergic neuroblastoma cell line sh sh5y - by Bioz Stars, 2024-05
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    dopaminergic human neuroblastoma cell line sh sy5y  (ATCC)


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    ATCC dopaminergic human neuroblastoma cell line sh sy5y
    Dopaminergic Human Neuroblastoma Cell Line Sh Sy5y, 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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    dopaminergic human neuroblastoma cell line sh sy5y  (ATCC)


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    ATCC dopaminergic human neuroblastoma cell line sh sy5y
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Dopaminergic Human Neuroblastoma Cell Line Sh Sy5y, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells"

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232315255

    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Expressing, Incubation, Immunofluorescence, Staining, Western Blot

    APM protects MPP + -induced mitochondria-dependent neurotoxicity in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h or 24 h. Expression of cleaved-Caspase3 and cleaved-PARP in rat embryo primary mesencephalic neurons ( A ) and SH-SY5Y cells ( B ) were detected by immunoblotting. βActin was used to confirm equal sample loading. Arrows: cleaved form. ( C ) SH-SY5Y cells were evaluated by fluorescence microscopy on the basis of morphological criteria after TUNEL staining (upper, scale bars: 100 μm) and JC-1 mitochondrial staining (lower, scale bars: 50 μm) and immunofluorescence positive cells were quantified by densitometric analysis ( D ). Nuclei were stained with DAPI. APM regulates mitochondrial apoptotic proteins ( E ). VDAC was used as mitochondrial loading control. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM protects MPP + -induced mitochondria-dependent neurotoxicity in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h or 24 h. Expression of cleaved-Caspase3 and cleaved-PARP in rat embryo primary mesencephalic neurons ( A ) and SH-SY5Y cells ( B ) were detected by immunoblotting. βActin was used to confirm equal sample loading. Arrows: cleaved form. ( C ) SH-SY5Y cells were evaluated by fluorescence microscopy on the basis of morphological criteria after TUNEL staining (upper, scale bars: 100 μm) and JC-1 mitochondrial staining (lower, scale bars: 50 μm) and immunofluorescence positive cells were quantified by densitometric analysis ( D ). Nuclei were stained with DAPI. APM regulates mitochondrial apoptotic proteins ( E ). VDAC was used as mitochondrial loading control. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Expressing, Western Blot, Fluorescence, Microscopy, TUNEL Assay, Staining, Immunofluorescence

    APM inhibited MPP + -induced SK channels in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM or BAPTA-AM (5 μM) for 1 h and then treated with MPP + for 24 h. ( A ) Intracellular Ca 2+ overload positive cells of rat embryo primary mesencephalic neurons were evaluated using fluorescence microscopy using FLUOFORTE staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). APM significantly inhibit MPP + -induced Kca2.2 and pCaMKⅡ expression in rat embryo primary mesencephalic neurons ( B ) and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM inhibited MPP + -induced SK channels in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM or BAPTA-AM (5 μM) for 1 h and then treated with MPP + for 24 h. ( A ) Intracellular Ca 2+ overload positive cells of rat embryo primary mesencephalic neurons were evaluated using fluorescence microscopy using FLUOFORTE staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). APM significantly inhibit MPP + -induced Kca2.2 and pCaMKⅡ expression in rat embryo primary mesencephalic neurons ( B ) and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Fluorescence, Microscopy, Staining, Expressing, Western Blot, Immunofluorescence

    APM alleviated MPP + -induced neuroinflammatory response, oxidative stress, and ER stress. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. Expression of inflammatory cytokines ( A ), HIF1α, NOX2, GRP78, and CHOP ( B ) in SH-SY5Y cells were analyzed by immunoblotting. ( C ) Expression of TNFα, NOX2, and CHOP in rat embryo primary mesencephalic neurons were confirmed by immunoblotting. βActin was used to confirm equal sample loading. ( D ) ROS generation in rat embryo primary mesencephalic neurons was detected by DCF-DA staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM alleviated MPP + -induced neuroinflammatory response, oxidative stress, and ER stress. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. Expression of inflammatory cytokines ( A ), HIF1α, NOX2, GRP78, and CHOP ( B ) in SH-SY5Y cells were analyzed by immunoblotting. ( C ) Expression of TNFα, NOX2, and CHOP in rat embryo primary mesencephalic neurons were confirmed by immunoblotting. βActin was used to confirm equal sample loading. ( D ) ROS generation in rat embryo primary mesencephalic neurons was detected by DCF-DA staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Expressing, Western Blot, Staining, Immunofluorescence

    APM strongly inhibited MPP + -induced phosphorylation of MAPK-ERK/p65/STAT3 signaling pathway. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h. Phosphorylation of ERK in SH-SY5Y cells ( A ) and rat embryo primary mesencephalic neurons ( B ) were analyzed by immunoblotting. MPP + -induced NFκB-p65 and STAT3 translocation in SH-SY5Y cells ( C ) and rat embryo primary mesencephalic neurons ( D ) were analyzed by immunoblotting. βActin was used to confirm equal sample loading. ( E ) The DNA-binding activity of NFκB-p65 and STAT3 in nuclear extracts was measured using EMSA. ( F ) Immunofluorescence double staining for TH (green) and pp65 (red) localization, and TH (green) and pSTAT3 (red) localization, in rat embryo primary mesencephalic neurons. Scale bars: 50 μm. Nuclei were stained with DAPI (blue). Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Figure Legend Snippet: APM strongly inhibited MPP + -induced phosphorylation of MAPK-ERK/p65/STAT3 signaling pathway. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h. Phosphorylation of ERK in SH-SY5Y cells ( A ) and rat embryo primary mesencephalic neurons ( B ) were analyzed by immunoblotting. MPP + -induced NFκB-p65 and STAT3 translocation in SH-SY5Y cells ( C ) and rat embryo primary mesencephalic neurons ( D ) were analyzed by immunoblotting. βActin was used to confirm equal sample loading. ( E ) The DNA-binding activity of NFκB-p65 and STAT3 in nuclear extracts was measured using EMSA. ( F ) Immunofluorescence double staining for TH (green) and pp65 (red) localization, and TH (green) and pSTAT3 (red) localization, in rat embryo primary mesencephalic neurons. Scale bars: 50 μm. Nuclei were stained with DAPI (blue). Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Techniques Used: Incubation, Western Blot, Translocation Assay, Binding Assay, Activity Assay, Immunofluorescence, Double Staining, Staining

    dopaminergic neuroblastoma cell lines  (ATCC)


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    ATCC dopaminergic human neuroblastoma cell line sh sy5y
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Dopaminergic Human Neuroblastoma Cell Line Sh Sy5y, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dopaminergic human neuroblastoma cell line sh sy5y/product/ATCC
    Average 99 stars, based on 1 article reviews
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    ATCC human dopaminergic neuroblastoma cell line sh sh5y
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Human Dopaminergic Neuroblastoma Cell Line Sh Sh5y, 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
    https://www.bioz.com/result/human dopaminergic neuroblastoma cell line sh sh5y/product/ATCC
    Average 86 stars, based on 1 article reviews
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    ATCC human dopaminergic neuroblastoma sh sy5y cell line
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Human Dopaminergic Neuroblastoma Sh Sy5y Cell Line, 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
    https://www.bioz.com/result/human dopaminergic neuroblastoma sh sy5y cell line/product/ATCC
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    ATCC human dopaminergic neuroblastoma sh sy5y cell lines
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Human Dopaminergic Neuroblastoma Sh Sy5y Cell Lines, 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
    https://www.bioz.com/result/human dopaminergic neuroblastoma sh sy5y cell lines/product/ATCC
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    ATCC dopaminergic neuroblastoma cell lines
    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. <t>SH-SY5Y</t> cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.
    Dopaminergic Neuroblastoma Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Journal: International Journal of Molecular Sciences

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    doi: 10.3390/ijms232315255

    Figure Lengend Snippet: Effects of APM on MPP + -induced TH and αSYN expression in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. ( A ) The morphological changes, TH and αSYN expression in dopaminergic neurons after exposure to MPP + in the presence or absence of APM. Immunofluorescence staining for TH (green) and αSYN (red) localization. Cells were counterstained with DAPI (blue). Scale bars: 50 μm. APM strongly reduced expression of TH reduction and accumulation of αSYN in rat embryo primary mesencephalic neurons ( B ), and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Article Snippet: A dopaminergic human neuroblastoma cell line SH-SY5Y (America Tissue Culture Collection, CRL-2266; ATCC, Manassas, VA, USA), was cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco).

    Techniques: Expressing, Incubation, Immunofluorescence, Staining, Western Blot

    APM protects MPP + -induced mitochondria-dependent neurotoxicity in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h or 24 h. Expression of cleaved-Caspase3 and cleaved-PARP in rat embryo primary mesencephalic neurons ( A ) and SH-SY5Y cells ( B ) were detected by immunoblotting. βActin was used to confirm equal sample loading. Arrows: cleaved form. ( C ) SH-SY5Y cells were evaluated by fluorescence microscopy on the basis of morphological criteria after TUNEL staining (upper, scale bars: 100 μm) and JC-1 mitochondrial staining (lower, scale bars: 50 μm) and immunofluorescence positive cells were quantified by densitometric analysis ( D ). Nuclei were stained with DAPI. APM regulates mitochondrial apoptotic proteins ( E ). VDAC was used as mitochondrial loading control. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Journal: International Journal of Molecular Sciences

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    doi: 10.3390/ijms232315255

    Figure Lengend Snippet: APM protects MPP + -induced mitochondria-dependent neurotoxicity in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h or 24 h. Expression of cleaved-Caspase3 and cleaved-PARP in rat embryo primary mesencephalic neurons ( A ) and SH-SY5Y cells ( B ) were detected by immunoblotting. βActin was used to confirm equal sample loading. Arrows: cleaved form. ( C ) SH-SY5Y cells were evaluated by fluorescence microscopy on the basis of morphological criteria after TUNEL staining (upper, scale bars: 100 μm) and JC-1 mitochondrial staining (lower, scale bars: 50 μm) and immunofluorescence positive cells were quantified by densitometric analysis ( D ). Nuclei were stained with DAPI. APM regulates mitochondrial apoptotic proteins ( E ). VDAC was used as mitochondrial loading control. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Article Snippet: A dopaminergic human neuroblastoma cell line SH-SY5Y (America Tissue Culture Collection, CRL-2266; ATCC, Manassas, VA, USA), was cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco).

    Techniques: Incubation, Expressing, Western Blot, Fluorescence, Microscopy, TUNEL Assay, Staining, Immunofluorescence

    APM inhibited MPP + -induced SK channels in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM or BAPTA-AM (5 μM) for 1 h and then treated with MPP + for 24 h. ( A ) Intracellular Ca 2+ overload positive cells of rat embryo primary mesencephalic neurons were evaluated using fluorescence microscopy using FLUOFORTE staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). APM significantly inhibit MPP + -induced Kca2.2 and pCaMKⅡ expression in rat embryo primary mesencephalic neurons ( B ) and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 compared to normal control.

    Journal: International Journal of Molecular Sciences

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    doi: 10.3390/ijms232315255

    Figure Lengend Snippet: APM inhibited MPP + -induced SK channels in dopaminergic neuronal cells. SH-SY5Y cells and rat embryo primary mesencephalic neurons were incubated in the presence or absence of APM or BAPTA-AM (5 μM) for 1 h and then treated with MPP + for 24 h. ( A ) Intracellular Ca 2+ overload positive cells of rat embryo primary mesencephalic neurons were evaluated using fluorescence microscopy using FLUOFORTE staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). APM significantly inhibit MPP + -induced Kca2.2 and pCaMKⅡ expression in rat embryo primary mesencephalic neurons ( B ) and SH-SY5Y cells ( C ). βActin was used to confirm equal sample loading. Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 compared to normal control.

    Article Snippet: A dopaminergic human neuroblastoma cell line SH-SY5Y (America Tissue Culture Collection, CRL-2266; ATCC, Manassas, VA, USA), was cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco).

    Techniques: Incubation, Fluorescence, Microscopy, Staining, Expressing, Western Blot, Immunofluorescence

    APM alleviated MPP + -induced neuroinflammatory response, oxidative stress, and ER stress. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. Expression of inflammatory cytokines ( A ), HIF1α, NOX2, GRP78, and CHOP ( B ) in SH-SY5Y cells were analyzed by immunoblotting. ( C ) Expression of TNFα, NOX2, and CHOP in rat embryo primary mesencephalic neurons were confirmed by immunoblotting. βActin was used to confirm equal sample loading. ( D ) ROS generation in rat embryo primary mesencephalic neurons was detected by DCF-DA staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Journal: International Journal of Molecular Sciences

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    doi: 10.3390/ijms232315255

    Figure Lengend Snippet: APM alleviated MPP + -induced neuroinflammatory response, oxidative stress, and ER stress. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 24 h. Expression of inflammatory cytokines ( A ), HIF1α, NOX2, GRP78, and CHOP ( B ) in SH-SY5Y cells were analyzed by immunoblotting. ( C ) Expression of TNFα, NOX2, and CHOP in rat embryo primary mesencephalic neurons were confirmed by immunoblotting. βActin was used to confirm equal sample loading. ( D ) ROS generation in rat embryo primary mesencephalic neurons was detected by DCF-DA staining (green, scale bars: 100 μm). Nuclei were stained with DAPI (blue). Immunoblotting and immunofluorescence positive cells were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Article Snippet: A dopaminergic human neuroblastoma cell line SH-SY5Y (America Tissue Culture Collection, CRL-2266; ATCC, Manassas, VA, USA), was cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco).

    Techniques: Incubation, Expressing, Western Blot, Staining, Immunofluorescence

    APM strongly inhibited MPP + -induced phosphorylation of MAPK-ERK/p65/STAT3 signaling pathway. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h. Phosphorylation of ERK in SH-SY5Y cells ( A ) and rat embryo primary mesencephalic neurons ( B ) were analyzed by immunoblotting. MPP + -induced NFκB-p65 and STAT3 translocation in SH-SY5Y cells ( C ) and rat embryo primary mesencephalic neurons ( D ) were analyzed by immunoblotting. βActin was used to confirm equal sample loading. ( E ) The DNA-binding activity of NFκB-p65 and STAT3 in nuclear extracts was measured using EMSA. ( F ) Immunofluorescence double staining for TH (green) and pp65 (red) localization, and TH (green) and pSTAT3 (red) localization, in rat embryo primary mesencephalic neurons. Scale bars: 50 μm. Nuclei were stained with DAPI (blue). Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Journal: International Journal of Molecular Sciences

    Article Title: Effects of Apamin on MPP + -Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells

    doi: 10.3390/ijms232315255

    Figure Lengend Snippet: APM strongly inhibited MPP + -induced phosphorylation of MAPK-ERK/p65/STAT3 signaling pathway. Dopaminergic neuronal cells were incubated in the presence or absence of APM for 1 h and then treated with MPP + for 12 h. Phosphorylation of ERK in SH-SY5Y cells ( A ) and rat embryo primary mesencephalic neurons ( B ) were analyzed by immunoblotting. MPP + -induced NFκB-p65 and STAT3 translocation in SH-SY5Y cells ( C ) and rat embryo primary mesencephalic neurons ( D ) were analyzed by immunoblotting. βActin was used to confirm equal sample loading. ( E ) The DNA-binding activity of NFκB-p65 and STAT3 in nuclear extracts was measured using EMSA. ( F ) Immunofluorescence double staining for TH (green) and pp65 (red) localization, and TH (green) and pSTAT3 (red) localization, in rat embryo primary mesencephalic neurons. Scale bars: 50 μm. Nuclei were stained with DAPI (blue). Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM. Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal control.

    Article Snippet: A dopaminergic human neuroblastoma cell line SH-SY5Y (America Tissue Culture Collection, CRL-2266; ATCC, Manassas, VA, USA), was cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco).

    Techniques: Incubation, Western Blot, Translocation Assay, Binding Assay, Activity Assay, Immunofluorescence, Double Staining, Staining