mouse anti ngf neutralizing antibodies  (Alomone Labs)


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    Alomone Labs mouse anti ngf neutralizing antibodies
    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of <t>NGF</t> before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag <t>MFG-E8</t> D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P
    Mouse Anti Ngf Neutralizing Antibodies, supplied by Alomone Labs, 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/mouse anti ngf neutralizing antibodies/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti ngf neutralizing antibodies - by Bioz Stars, 2022-01
    86/100 stars

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    1) Product Images from "Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration"

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1155-z

    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P
    Figure Legend Snippet: Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Techniques Used: Activity Assay, Cell Culture, Staining

    NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P
    Figure Legend Snippet: NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Techniques Used: Cell Culture, Staining

    PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P
    Figure Legend Snippet: PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Techniques Used: Cell Culture, In Vitro, Staining

    Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p
    Figure Legend Snippet: Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Techniques Used: Cell Culture, Purification, Labeling

    Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition
    Figure Legend Snippet: Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Techniques Used: Blocking Assay, Cell Culture, Staining

    Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P
    Figure Legend Snippet: Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Techniques Used: Cell Culture, Staining

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    Alomone Labs mouse anti ngf neutralizing antibodies
    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of <t>NGF</t> before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag <t>MFG-E8</t> D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P
    Mouse Anti Ngf Neutralizing Antibodies, supplied by Alomone Labs, 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/mouse anti ngf neutralizing antibodies/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti ngf neutralizing antibodies - by Bioz Stars, 2022-01
    86/100 stars
      Buy from Supplier

    92
    Alomone Labs monoclonal anti ngf antibody
    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of <t>NGF</t> before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag <t>MFG-E8</t> D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P
    Monoclonal Anti Ngf Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti ngf antibody/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti ngf antibody - by Bioz Stars, 2022-01
    92/100 stars
      Buy from Supplier

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    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Activity Assay, Cell Culture, Staining

    NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Staining

    PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, In Vitro, Staining

    Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Purification, Labeling

    Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Blocking Assay, Cell Culture, Staining

    Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Staining