human ngf neutralizing antibody  (R&D Systems)

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: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Neuronal EVs promote neural induction and cell proliferation of mESCs. (A) Gene expression analysis of Nestin and Six3 in mESCs treated without (control) or with indicated EVs. EV number was quantified by Nanosight 2000. Data plotted were from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P < 0.05). (B) Gene expression analysis of Nestin and Six3 in mESCs treated for 4 d without (control) or with different doses of N6-EV. Data plotted were from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). (C) Gene expression analysis of Sox1 , Pax6 , and Tuj1 in mESCs treated for 4 d with 2× 10 9 PC12-EV, N6-EV, or N6-EV together with NGF neutralizing antibody (500 ng/ml). The values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (D) Immunostaining of Nestin (green, Alexa Fluor 488) and Pax6 (red, Alexa Fluor 568) in mESCs as described in B. Scale bar, 25 µm. (E) Quantitative analysis of the percentage of cells containing indicated markers compared with DAPI-stained cells. The values represent the mean ± SD, from three independent experiments (*, P < 0.05). Error bars represent SD from independent samples. (F) Gene expression analysis of Sox1 , Pax6 , and Nestin in mESCs treated for 4 d with 2 × 10 9 EVs from undifferentiated pluripotent ESCs (ES-D0), EBs of ES differentiated for 8 d in KSR medium (ES-D8), and EBs trypsinized in N2 medium for an additional 4 d (ES-D12). The values represent the mean ± SD, from two independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (G) The cellular morphology of mESCs treated with PC12-Exo or different doses of N6-EV in N2B27 medium for 4 d. Scale bars, 200 µm. (H) Quantitative analysis of mESC number treated with or without N6-EV. Data plotted were from two independent experiments. The values represent the mean ± SD (*, P < 0.05). Error bars represent SD from independent samples. (I) Proliferation analysis of mESCs with BrdU staining after EV treatment. The values represent the mean ± SD, from two independent experiments (*, P < 0.05). Error bars represent SD from independent samples.

Article Snippet: The following antibodies were used: rabbit anti-cyclin D1, anti-cyclin A2, anti-CDK4, anti-CD9, anti-Flotillin 2, anti-p21, anti-p27, anti-p57, anti-nucleolin, anti-Ngn2, anti-integrin (ab134175, ab181591, ab199728, ab92726, ab96507, ab109199, ab32034, ab75974, ab129200, ab109236, and ab131055; Abcam), rabbit anti-CDK6 (GTX103992; GeneTex), rabbit anti–cyclin B1, anti–cyclin E1, anti–phospho-Rb (4138T, 20808S, and 9307; Cell Signaling Technology), rabbit anti-GFP (NC9589665; Fisher Scientific), rabbit anti-Lin28 (11724-1-AP; Proteintech); mouse anti-actin (ab8224; Abcam), mouse anti–cyclin D2, anti–cyclin D3, anti-Alix, anti-CD81 (sc-376676, sc-6283, sc-53540, and sc-166029; Santa Cruz Biotechnology), mouse anti-GM130 (610823; BD Biosciences), mouse anti-Tsg101 (GTX70255; GeneTex); rat anti-CD63 (clone R5G2, LS-C179520; LSBIO), rat anti-Hsc70 (ab19136; Abcam), HRP-conjugated streptavidin (N100; Thermo Fisher Scientific), GFP protein recombinant (PRO-687; ProSpec), NGF neutralizing antibody (MAB256-100; R&D Systems), HRP-linked IgG from mouse and rabbit (NXA931,45000682; Fisher Scientific), and HRP-linked IgG from rat (A5795; Sigma-Aldrich).

Techniques: Gene Expression, Control, Immunostaining, Staining, BrdU Staining

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: RA-induced EVs promote mESC neural fate commitment. (A) The cellular morphology of PC12 cells untreated or treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 µm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells untreated and treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (C) Gene expression analysis of mESCs treated with EVs purified from N2A cells (N2A-EV) or EVs purified from 6-d RA-treated cells (RA-EV). The values represent the mean ± SD, from two independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (D) Gene expression of Six3 and Pax6 in mESCs treated with different doses of RA-EV. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). Error bars represent SD from independent samples. (E) Immunostaining of mESCs described in A. Cells were stained with Nestin (green, Alexa Fluor 488) and Pax6 antibodies (red, Alexa Fluor 568). Scale bars, 25 µm. (F) Quantitative analysis of the staining in C. The values represent the mean ± SD, from three independent experiments (*, P < 0.05). Error bars represent SD from independent samples.

Article Snippet: The following antibodies were used: rabbit anti-cyclin D1, anti-cyclin A2, anti-CDK4, anti-CD9, anti-Flotillin 2, anti-p21, anti-p27, anti-p57, anti-nucleolin, anti-Ngn2, anti-integrin (ab134175, ab181591, ab199728, ab92726, ab96507, ab109199, ab32034, ab75974, ab129200, ab109236, and ab131055; Abcam), rabbit anti-CDK6 (GTX103992; GeneTex), rabbit anti–cyclin B1, anti–cyclin E1, anti–phospho-Rb (4138T, 20808S, and 9307; Cell Signaling Technology), rabbit anti-GFP (NC9589665; Fisher Scientific), rabbit anti-Lin28 (11724-1-AP; Proteintech); mouse anti-actin (ab8224; Abcam), mouse anti–cyclin D2, anti–cyclin D3, anti-Alix, anti-CD81 (sc-376676, sc-6283, sc-53540, and sc-166029; Santa Cruz Biotechnology), mouse anti-GM130 (610823; BD Biosciences), mouse anti-Tsg101 (GTX70255; GeneTex); rat anti-CD63 (clone R5G2, LS-C179520; LSBIO), rat anti-Hsc70 (ab19136; Abcam), HRP-conjugated streptavidin (N100; Thermo Fisher Scientific), GFP protein recombinant (PRO-687; ProSpec), NGF neutralizing antibody (MAB256-100; R&D Systems), HRP-linked IgG from mouse and rabbit (NXA931,45000682; Fisher Scientific), and HRP-linked IgG from rat (A5795; Sigma-Aldrich).

Techniques: Gene Expression, Purification, Immunostaining, Staining