rat beta ngf duoset elisa kit  (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: ELISA with supernatants from neuronally differentiated ADSCs. Expression of NGF is especially upregulated after differentiation with Protocol 3. The obtained values were not normalized to the total cell number.

Article Snippet: The rat NGF Duo Set ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the GDNF (Rat) ELISA Kit (Abnova, Taipei, Taiwan) were used, according to the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

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: ELISA: expression of NGF after differentiation with Protocols 1, 2, and 3. The obtained values were not normalized to the total cell number.

Article Snippet: The rat NGF Duo Set ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the GDNF (Rat) ELISA Kit (Abnova, Taipei, Taiwan) were used, according to the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: NGF secretion by human pulmonary arterial cells after treatment with IL-1β or H 2 O 2. NGF secretion by human pulmonary arterial smooth muscle cells (hPASMC, ( a , b )) or by human pulmonary arterial endothelial cells (hPAEC, ( c , d )) was assessed in the absence or presence of interleukin-1β (IL-1β, 0.1–100 ng/mL, 24 h, ( a , c )) or hydrogen peroxide (H 2 O 2 , 0–50 µM, 24 h, ( b , d )). NGF secretion was determined by ELISA (results expressed as pg NGF/µg total proteins). The data represent the means ± SEM of n = 5 independent experiments performed in triplicate on cells from three to five control donors. *: p < 0.05 and **: p < 0.01 versus untreated control cells.

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: TGF-β1 secretion by human pulmonary arterial cells after treatment with IL-1β or H 2 O 2 , and TGF-β1 ability to trigger NGF secretion by these cells. TGF-β1 secretion by human pulmonary arterial smooth muscle cells (hPASMC, ( a )) or human pulmonary arterial endothelial cells (hPAEC, ( c )) was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h) or hydrogen peroxide (H 2 O 2 , 10 µM, 24 h). NGF secretion by ( b ) hPASMC or ( d ) hPAEC was assessed in the absence or presence of TGF-β1 (0.1–10 ng/mL, 24 h). NGF and TGF-β1 secretions were determined by ELISA (results expressed as pg TGF-β1 or pg NGF/µg total proteins). The data represent the means ± SEM of n = 5 independent experiments performed in triplicate on cells from three to five control donors. * p < 0.05 and ** p < 0.01 versus untreated control cells (Contr or 0 ng/mL of TGF-β1).

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: Control of TGF-β1 decreased secretion after transfection of pulmonary arterial cells with an anti TGF-β1 siRNA. TGF-β1 secretion by human pulmonary arterial smooth muscle cells (hPASMC, ( a )) or human pulmonary arterial endothelial cells (hPAEC, ( b )) was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h) or hydrogen peroxide (H 2 O 2 , 10 µM, 24 h). Cells were pre-treated for 48 h with an anti-TGF-β1 siRNA (SiTGF, 1 nM). The effect of this anti-TGF-β1 siRNA on TGF-β1 basal secretion was evaluated. Furthermore, the effects of the transfecting agent INTERFERin ® (Interf) alone and of a non-relevant scramble siRNA (SiScrbl, 1 nM) on TGF-β1 basal and induced secretions were evaluated. TGF-β1 secretion was determined by ELISA (results expressed as pg TGF-β1/µg total proteins). The data represent the means ± SEM of n = 5 independent experiments performed in triplicate on cells from three to five donors. * p < 0.05 and ** p < 0.01 versus untreated control cells (Contr). # p < 0.05 and ## p < 0.01 versus cells treated with either IL-1β or H 2 O 2 .

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Control, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: Role of TGF-β1 in IL-1β- or H 2 O 2 -induced increase in NGF secretion by human pulmonary arterial cells. NGF secretion by human pulmonary arterial smooth muscle cells (hPASMC, ( a , b )) or human pulmonary arterial endothelial cells (hPAEC, ( c , d )) was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h) or hydrogen peroxide (H 2 O 2 , 10 µM, 24 h). Involvement of the transforming growth factor-β1 (TGF-β1) was investigated in ( a ) hPASMC or ( c ) hPAEC pre-treated for 48 h with an anti-TGF-β1 siRNA (SiTGF, 1 nM). Effect of this anti-TGF-β1 siRNA on NGF basal secretion was evaluated. Furthermore, the effects of the transfecting agent INTERFERin ® (Interf) alone and of a non-relevant scramble siRNA (SiScrbl, 1 nM) on NGF basal and induced secretions were evaluated. TGF-β1 involvement was also investigated through pre-treatment (30 min) of ( b ) hPASMC or ( d ) hPAEC with anti-TGF-β1 blocking antibodies (AbTGF, 1 µg/mL). Effect of these antibodies on NGF basal secretion was evaluated. NGF secretion was determined by ELISA (results expressed as pg NGF/µg total proteins). The data represent the means ± SEM of n = 5 independent experiments performed in triplicate on cells from three to five control donors. * p < 0.05 and ** p < 0.01 versus untreated control cells (Contr). # p < 0.05 and ## p < 0.01 versus cells treated with either IL-1β or H 2 O 2 .

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: Expression of the TGF-β1 receptor TβRI in human pulmonary arterial cells and its role in IL-1β- or H 2 O 2 -induced increase in NGF secretion by these cells. ( a ) Basal protein expression of TβRI was assessed by Western blotting analysis in human pulmonary arterial smooth muscle cells (hPASMC) or human pulmonary arterial endothelial cells (hPAEC). Immunoblots presented on cells from two control donors (D1 and D2) are representative of experiments conducted on pulmonary arterial cells from n = 3–5 control donors, showing identical results. Results are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ( b , c ) NGF secretion by ( b ) hPASMC or ( c ) hPAEC was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h) or hydrogen peroxide (H 2 O 2 , 10 µM, 24 h). Involvement of TβRI was investigated through pre-treatment (30 min) of ( b ) hPASMC or ( c ) hPAEC with SB525334, an inhibitor of TβRI receptor kinase activity (SB, 1 µM). The effect of this inhibitor on NGF basal secretion was investigated. In addition, the effects of dimethyl sulfoxide (DMSO), the vehicle of the SB compound, on NGF basal and induced secretions were evaluated. NGF secretion was determined by ELISA (results expressed as pg NGF/µg total proteins). The data represent the means ± SEM of n = 5 independent experiments performed in triplicate on cells from three to five donors. * p < 0.05 and ** p < 0.01 versus untreated control cells (Contr). # p < 0.05 versus cells treated with IL-1β or H 2 O 2 .

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Expressing, Western Blot, Control, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: Signaling pathways activated by the TGF-β1 receptor TβRI in human pulmonary arterial cells to participate in the IL-1β- or H 2 O 2 -induced increase in NGF secretion by these cells. ( a – f ) NGF secretion by human pulmonary arterial smooth muscle cells (hPASMC, ( a – c )) or human pulmonary arterial endothelial cells (hPAEC, ( d – f )) was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h), hydrogen peroxide (H 2 O 2 , 10 µM, 24 h), or TGF-β1 (5 ng/mL, 24 h). Signaling pathways involved were investigated through cell pre-treatment (45 min) with the p38 inhibitor SB203880 (+ SB2, 2 µM) or with the Smad3 inhibitor SIS3 (+SIS3, 10 µM). NGF secretion was determined by ELISA (results expressed as pg NGF/µg total proteins). The data represent the means ± SEM of n = 3 independent experiments performed in triplicate on cells from three control donors. * p < 0.05 and ** p < 0.01 versus untreated control cells (Contr). ( g,h ) Time-course of TGF-β1-induced phosphorylation of Smad3 and p38 in ( g ) hPASMC or ( h ) hPAEC. Cells were incubated with TGF-β1 (5 ng/mL) for 10–60 min, and Western blotting analysis used anti-phospho-Smad3 or anti-phospho-p38 antibodies, as well as anti-Smad3 and anti-p38 antibodies as controls. The immunoblots presented are representative of experiments conducted on pulmonary arterial cells from n = 2–4 control donors, showing identical results.

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Protein-Protein interactions, Enzyme-linked Immunosorbent Assay, Control, Phospho-proteomics, Incubation, Western Blot

Journal: Cells

Article Title: Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism

doi: 10.3390/cells11182795

Figure Lengend Snippet: Role of TGF-β1 in IL-1β- or H 2 O 2 -induced increase in NGF secretion by rat whole pulmonary arteries. ( a ) NGF secretion by rat whole pulmonary arteries was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h), hydrogen peroxide (H 2 O 2 , 10 µM, 24 h), or transforming growth factor-β1 (TGF-β1, 5 ng/mL, 24 h). Involvement of TGF-β1 in IL-1β- or H 2 O 2 -induced NGF secretion was investigated through pre-treatment (30 min) of pulmonary arteries with anti-TGF-β1 blocking antibodies (AbTGF, 1 µg/mL). The effect of these antibodies on NGF basal secretion was evaluated. NGF secretion was determined by ELISA (results expressed as pg NGF/µg total proteins). The data represent the means ± SEM with experiments conducted on pulmonary arteries of n = 5 rats per group. * p < 0.05 versus untreated pulmonary arteries (Contr). # p < 0.05 versus pulmonary arteries treated with either IL-1β or H 2 O 2 . ( b ) TGF-β1 secretion by rat whole pulmonary arteries was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h) or hydrogen peroxide (H 2 O 2 , 10 µM, 24 h). TGF-β1 secretion was determined by ELISA (results expressed as pg TGF-β1/µg total proteins). The data represent the means ± SEM with experiments conducted on pulmonary arteries of n = 5 rats per group. * p < 0.05 and *** p < 0.001 versus untreated pulmonary arteries (Control). ( c ) Basal protein expression of TβRI was assessed by Western blotting analysis in rat whole pulmonary arteries. The immunoblots presented were performed on pulmonary arteries from five different rats (rats 1–5). Results are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ( d ) NGF secretion by rat whole pulmonary arteries was assessed in the absence or presence of interleukin-1β (IL-1β, 10 ng/mL, 24 h) or hydrogen peroxide (H 2 O 2 , 10 µM, 24 h). Involvement of TβRI was investigated through pre-treatment (30 min) of pulmonary arteries with SB525334, an inhibitor of TβRI receptor kinase activity (SB, 1 µM). The effect of this inhibitor on NGF basal secretion was investigated. In addition, the effects of dimethyl sulfoxide (DMSO), the vehicle of the SB compound, on NGF basal and induced secretions were evaluated. NGF secretion was determined by ELISA (results expressed as pg NGF/µg total proteins). The data represent the means ± SEM with experiments conducted on pulmonary arteries of n = 5 rats per group. * p < 0.05 versus untreated pulmonary arteries (Contr). # p < 0.05 versus pulmonary arteries treated with IL-1β or H 2 O 2 .

Article Snippet: Determination of NGF or TGF-β1 levels were assessed by use of ELISA kits, according to the manufacturer’s instructions (for NGF rat samples: Rat β-NGF ELISA Kit from Millipore; for NGF human samples: NGF Rapid Kit ELISA Human from Biosensis, Thebarton, Australia; and for TGF-β1 rat and human samples: TGF-β1 E max Immunoassay system from Promega, Charbonnières-les-Bains, France).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Control, Expressing, Western Blot, Activity Assay