neutralizing anti-ngf antibody  (Millipore)

Journal: Cells

Article Title: Peripheral Nerve Regeneration–Adipose-Tissue-Derived Stem Cells Differentiated by a Three-Step Protocol Promote Neurite Elongation via NGF Secretion

doi: 10.3390/cells11182887

Figure Lengend Snippet: Lengths of neurites in µm on NG108-15 cells after cultivation with conditioned Protocol-3 medium, with or without NGF-neutralizing antibodies. The mean value and standard error of the mean for each condition are shown. ***: p -value < 0.001.

Article Snippet: In order to verify the results, NG108-15 cells were also incubated with either 100 ng/mL recombinant NGF (PeproTech) or a 1:20,000 dilution of the neutralizing anti-NGF antibody (Sigma-Aldrich).

Techniques:

Journal: Cell reports

Article Title: RIP2 Gates TRAF6 Interaction with Death Receptor p75 NTR to Regulate Cerebellar Granule Neuron Survival.

doi: 10.1016/j.celrep.2018.06.098

Figure Lengend Snippet: Figure 1. Expression of p75NTR, NGF, and RIP2 during Postnatal Cerebellar Develop- ment (A–C) p75NTR mRNA (A), Ngf mRNA (B), and ripk2 mRNA (C) quantification relative to 18S RNA levels in cerebellum at the indicated postnatal (P) ages (mean ± SEM of data from 5 cerebella at each age). (D–F) Representative immunoblots and quantifi- cation of p75NTR (D) mature (mNGF), proNGF (E), and RIP2 (F) protein in total cerebellar protein extracts at the indicated ages (mean ± SEM densitometry from six experiments; representative blots are shown). (G) Micrographs of representative P7 cerebellum sections labeled with RIP2 antisense in situ hybridization probe, anti-p75NTR antibody, and counterstained with DAPI. The scale bars repre- sent 50 mm. (H) Immunoblots showing the expression of p75NTR and RIP2 in CGNs cultured for the indi- cated days in vitro (DIV).

Article Snippet: Mature NGF (N-100), brain-derived neurotrophic factor (BDNF) (B-250), and anti-NGF/ proNGF (total NGF) neutralizing antibody (ALM-006) were obtained from Alomone.

Techniques: Expressing, Western Blot, Labeling, In Situ Hybridization, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: a. Schematic representation of NGF deprivation paradigm in microfluidic devices. Cell bodies (CB) and distal axons (DA) are separated. For NGF deprivation, sympathetic neurons were maintained in NGF-deficient media containing 1µg/mL anti-NGF antibody. b. Representative images of β3-tubulin immuno-stained distal sympathetic axons before treatment (0hr), 12, 24 and 36 hours after NGF deprivation. Scale bar = 50µm. c. Degeneration time course after NGF deprivation. Latent and catastrophic phases of degeneration are noted. Nonlinear regression curves drawn according to Hill equation. n=3 for each time point. Data points represent as mean±SEM. d. Fluo4-AM calcium imaging of sympathetic axons at indicated times after NGF deprivation. Yellow box indicates individual axon as region of interest. Red box indicates axonal spheroid as region of interest. White arrowheads indicate the formation and growth of spheroid after 17 hours of NGF deprivation. Scale bar = 10µm. e. Calcium fluorescence change of control or NGF deprived axons over time. For the “NGF deprivation” condition, neurons were deprived of NGF for 17 hours, and then incubated with Fluo4-AM for calcium imaging. For the “Control” condition, no NGF deprivation was performed. Grey vertical dotted line indicates the onset of NGF deprivation induced catastrophic phase (18 hours after NGF deprivation). Black horizontal dotted line indicates the baseline without any calcium change. Total number of n=8 (NGF deprivation) and n=18 (control) of axons from 3 independent litters were quantified. Significant difference is determined by two-way ANOVA with multiple comparisons, *p<0.05, ***p<0.0001. Data shown as mean±SEM. f. Calcium fluorescence and size change of axonal spheroid after 17 hours of NGF deprivation. Total number of n=18 axonal spheroids from 3 independent litters were quantified. Data shown as mean±SEM. g. Quantification of axonal spheroid number per 100µm of axon at indicated times after NGF deprivation. Nonlinear regression curves were drawn according to Hill equation. Total number of n=11 axons from 3 independent litters were counted. Data shown as mean±SEM.

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Staining, Imaging, Fluorescence, Control, Incubation

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: a. Fluo4-AM calcium imaging of sympathetic axons after 18 hours of NGF deprivation in the presence of DMSO, 50µM V-ZAD-FMK, 20µM Calpain inhibitor III, and 1µg/mL Actinomycin, respectively. Scale bar = 10µm. b. Quantification of axonal spheroid number per 100µm of axons treated with DMSO (black), 50µM V-ZAD-FMK (red), 20µM Calpain inhibitor III (blue), or 1µg/mL Actinomycin (magenta) at indicated times after NGF deprivation. Nonlinear regression curves were drawn according to Hill equation. Total number of n=26 (DMSO), n=25 (Z-VAD-FMK), n=20 (Calpain inhibitor III), n=17 (Actinomycin) axons from 3 independent litters were counted. Data shown as mean±SEM. c. Representative images of axonal spheroids after 18 hours of NGF deprivation. Fluo4-AM (green) indicates intra-axonal calcium, and Annexin V (magenta) indicates exposure of phosphatidylserine on the extracellular surface of axonal spheroids. Scale bar = 5µm. d. Quantification of the percentage of fluorescent Annexin V positive spheroids after 17 to 19.5 hours of NGF deprivation. Total number of n=15 axons from 3 independent litters were counted. Data shown as mean±SEM.

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Imaging

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: a. Schematic representation of the experimental paradigm to assess membrane rupture model using fluorescent dextran. At 17 hours and 20 minutes of NGF deprivation, fluorescent dextran (red) is not taken up by the axon (black, negative space). However, by 18 hours of NGF deprivation, as the plasma membrane loses integrity and ruptures, fluorescent dextran (red) can diffuse into spheroids turning them red. Spheroids with intact membrane remain black. b. Representative images of dextran 3 kDa (red) entry to axonal spheroids (black) from 17 hours and 20 minutes to 18 hours and 30 minutes of NGF deprivation (left column), and dextran exclusion in untreated axons (right column). White arrowheads indicate that dextran 3 kDa enter to axonal spheroids after 18 hours of NGF deprivation. Scale bar = 10µm. c. Quantification of the percentages of fluorescent 3 kDa (red), 10 kDa (magenta), and 70 kDa (blue) dextran positive spheroids 20 to 90 minutes after 17 hours NGF deprivation. Black line (control) indicates the percentages of fluorescent 3 kDa dextran positive spheroids in the presence of NGF. Total number of n=15 (3 kDa), n=15 (10 kDa), n=12 (70 kDa), and n=9 (control) axons from 3 independent litters were counted. Data shown as mean±SEM. d. Measurement of extracellular calcium expelled from axons into calcium free, FBS free media. All axons were grow in regular DMEM media then switched to calcium free, FBS free media prior to NGF deprivation. In the “Control CM” group, media was collected from axons grown in the presence of NGF. In “NDCM” groups, medium were collected at 12, 18, and 24 hours after NGF deprivation. Values were analyzed from n=7 (Control CM), n=5 (12hrs NDCM), n=6 (18hrs NDCM), and n=7 (24hrs NDCM) independent replicates. Significance is determined by one-way ANOVA with multiple comparisons, **p<0.001. Data shown as mean±SEM. e. Measurement of extracellular calcium extruded from axons into FBS free, IS21 supplemented DMEM media with or without 18 hours of NGF deprivation. All axons were grow in FBS free, IS21 supplemented media prior to NGF deprivation. Values were analyzed from n=8 (Control CM) and n=3 (18hrs NDCM) independent replicates. Significance is determined by unpaired t-test, *p<0.05. Data shown as mean±SEM.

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Membrane, Clinical Proteomics, Control

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: a. Conditioned media degeneration paradigm. WT sympathetic neurons were globally deprived of NGF for 12 hours followed by addition of conditioned media to the axons for 5 hours. NGF deprivation conditioned media (NDCM) was collected from degenerated axons after 24 hours of NGF deprivation. b. Representative images of β3-tubulin immuno-stained distal sympathetic axons after treatment with Control CM and NDCM for 5 hours in the presence and absence of NGF. Scale bar = 50 µm. c. Quantification of (b). Values are represented as mean±SEM. Significant difference is determined by two-way ANOVA with multiple comparisons, n.s.=not significant, ***p<0.0001. n=4 or more for each group. For each repeat, at least 100 axons were scored for degeneration. d. Representative images of β3-tubulin immunostained distal sympathetic axons after 24 and 48 hours of NGF deprivation in media with calcium (DMEM) and calcium free media. Axons were cultured in regular media (DMEM) and then switched to calcium free media at the time of NGF deprivation. Scale bar = 50 µm. e. Quantification of (d). Values are represented as mean±SEM. Significant difference is determined by two-way ANOVA with multiple comparisons, n.s.=not significant. ***p<0.0001, n=3 for each group. For each repeat, at least 100 axons were scored for degeneration. f. Representative images of β3-tubulin immunostained distal sympathetic axons after treatment with NDCM for 5 hours in the presence of DMSO, 50µM Z-VAD-FMK, and 20µM Calpain inhibitor III, respectively. All cultures were globally deprived of NGF for 12 hours prior to NDCM incubation. Scale bar = 50 µm. g. Quantification of (f). Values are represented as mean±SEM. Significant difference is determined by one-way ANOVA with multiple comparisons, n.s.=not significant, *p<0.05, n=7 or more for each group. For each repeat, at least 100 axons were scored for degeneration.

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Staining, Control, Cell Culture, Incubation

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: a. Fluo4-AM calcium imaging of wild-type, DR6 -/- and p75NTR -/- sympathetic axons 18 hours of NGF deprivation. Scale bar = 10µm. b,d. Spheroidal calcium fluorescence (b) and size change (d) of wild-type, DR6 -/- and p75NTR -/- sympathetic axons after indicated time of NGF deprivation. Black horizontal dotted line in (b) indicates the baseline without any calcium change. Lower x-axis labeled with blue correlates with time of NGF deprivation in p75NTR -/- sympathetic axons. Individual axonal spheroids were quantified: n=32 (wild-type), n=30 ( DR6 -/- ), and n=22 ( p75NTR -/- ) spheroids from cultured neurons harvested from 3 independent litters. Data shown as mean±SEM. c. Quantification of axonal spheroid number per 100µm of wild-type, DR6 -/- and p75NTR -/- sympathetic axon at indicated times after 17 hours of NGF deprivation. Individual axonal spheroids were counted: n=20 (wild-type), n=29 (( DR6 -/- ), and n=22 ( p75NTR -/- ) axons from cultured neurons harvested from 3 independent litters. Data shown as mean±SEM. e. Catastrophic axon degeneration paradigm to test the pro-degenerative effect of mutant NDCM. Wild-type sympathetic neurons were globally deprived of NGF for 12 hours followed by addition of conditioned media derived from mutant axons for 5 hours. Mutant NDCM was collected from degenerating p75NTR -/- or DR6 -/- axons 24 hours after NGF deprivation. f-g. Representative images of wild-type distal sympathetic axons immunostained for β3-tubulin after treatment with Control CM and NDCM collected from p75NTR -/- (f) and DR6 -/- axons (g) for 5 hours. Scale bar = 50 µm. h. Quantification of (f-g).Values are represented as mean±SEM. Left two columns represent percentages of degeneration of wild-type sympathetic axons treated with wild-type NDCM and Control CM, respectively. Significance is determined by two-way ANOVA with multiple comparisons, n.s.=not significant, *p<0.05, **p<0.001, ***p<0.0001. n=3 or more for each group. For each repeat, at least 100 axons were scored for degeneration.

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Imaging, Fluorescence, Labeling, Cell Culture, Mutagenesis, Derivative Assay, Control

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: a. Representative images of wild-type sympathetic axons immunostained for GST tag with or without NGF deprivation. All images except the first one show axons incubated with Rhotekin-RBD GST-fusion protein that binds active Rho proteins after fixation. Axon in the bottom image was NGF deprived and treated with 1µg/mL Rho inhibitor CT04 for 3 hours. Scale bar = 5 µm. b. Fluo4-AM calcium imaging of wild-type grown in NGF or NGF deprived sympathetic axons with or without drug treatment. For the “CT04” group, wild-type axons were incubated in SCG media containing 1µg/mL Rho inhibitor CT04, for 2 hours prior to 17 hours of NGF deprivation. For the “Cytochalasin D” group, wild-type axons were incubated in SCG media containing 10µg/mL actin polymerization inhibitor for 2 hours prior to 17 hours of NGF deprivation. Scale bar = 10µm. c. Quantification of axonal spheroid number per 100µm of wild-type sympathetic axons after 18 hours of NGF deprivation with or without CT04 or Cytochalasin D treatment. Total number of n=9 (Control), n=8 (CT04), n=22 (Cytochalasin D) axons from cultured neurons harvested from 3 independent litters were quantified. Significant difference is determined by ordinary one-way ANOVA with multiple comparisons, ***p<0.0001. Data shown as mean±SEM. d. Representative images of wild-type distal sympathetic axons immuno-stained for β3-tubulin with and without and 24 hours after NGF deprivation in the absence and presence of CT04 or Cytochalasin D. Scale bar = 50µm. e. Quantification of (d). Values are represented as mean±SEM. Significant difference is determined by two-way ANOVA with multiple comparisons, ***p<0.0001, n.s.=not significant, n=3 or more for each group. For each repeat, at least 100 axons were scored for degeneration. f. Fluo4-AM calcium imaging of p75NTR -/- sympathetic axons grown in the presence or absence of NGF with or without CN03 treatment. For “CN03” group, p75NTR -/- axons were incubated in SCG media containing 1µg/mL Rho activator CN03 for 2 hours before 17 hours of NGF deprivation. Scale bar = 10µm. g. Quantification of number of spheroids per 100µm of p75NTR -/- sympathetic axons after 18 hours of NGF deprivation in the absence and presence of CN03. Individual axons were counted: n=16 (Control) and n=12 (CN03) axons from 3 independent replicates. Significance is determined by unpaired t test, ***p<0.0001. Data shown as mean±SEM. h. Representative images of p75NTR -/- distal sympathetic axons immuno-stained for β3-tubulin after treatment with or without CN03 for 5 hours. All cell cultures were pre-treated with 12 hours of NGF deprivation. Scale bar = 50µm. i. Quantification of (h). Values are represented as mean±SEM. Significance is determined by unpaired t test, **p<0.001, n=7 (Control) and n=3 (CN03). For each repeat, at least 100 axons were scored for degeneration.

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Incubation, Imaging, Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture

doi: 10.1101/710111

Figure Lengend Snippet: After NGF deprivation, prodegenerative transcription is upregulated. Axoplasmic calcium is increased and enriched in spheroids prior to catastrophic phase. Spheroid formation is regulated by p75NTR, Rho activity, and caspase activation. The calcium electrochemical gradient across the membrane is disrupted by spheroidal rupture, which may also lead to the release of intra-axonal prodegenerative molecules to extracellular space, acting as extrinsic factors to promote degeneration in a paracrine or autocrine manner. We speculate that p75NTR plays important role in calcium dynamics during the latent phase, while DR6 can be activated by prodegenerative NDCM to mediate downstream catastrophic degeneration pathways (e.g. DLK/JNK, calpastatin, calpain).

Article Snippet: For NGF deprivation, cultures were washed three times with medium lacking NGF and then maintained in NGF-deficient media containing a neutralizing antibody (1μg/mL anti-NGF antibody, Millipore) through designated time points at 37°C.

Techniques: Activity Assay, Activation Assay, Membrane

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: 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 < 0.05, ** p < 0.01. Scale bar: 50 μm, N = 3 chambers per treatment

Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 0.1 mg/ml mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240).

Techniques: Cell Culture, In Vitro, Staining

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: 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 < 0.001. Scale bar: 50 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 0.1 mg/ml mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240).

Techniques: Cell Culture, Staining

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: 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 0.1 mg/ml mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240).

Techniques: Cell Culture, Staining

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: 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 < 0.001. 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 0.1 mg/ml mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240).

Techniques: Cell Culture, Staining

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: 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 < 0.05, ** P < 0.01, *** P < 0.001 (In H all p -values are compared to vehicle control). 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 0.1 mg/ml mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240).

Techniques: Cell Culture, Staining