Ngf elisa kit

In order to investigate the release of liposome under different pH, 71.6 g disodium hydrogen phosphate (Na₂HPO₄) and 31.2 g sodium dihydrogen phosphate (NaH₂PO₄) were dissolved in 1000 ml deionized water, respectively, and configured to obtain 0.2 M Na₂HPO₄ and NaH₂PO₄ mother liquor, and PBS solutions could be obtained via mixing the mother liquor according to Supplementary Table 3.
MSaP-aL/p was immersed in 50 ml centrifuge tubes containing 10 ml pH 7.4, pH 6.6, and pH 5.8 PBS, respectively. All centrifugal tubes were placed at 37 °C, on the 120 cycles min⁻¹ constant temperature vibrator (Thermo, USA). After 3, 6, 9, 12, 15, 18, 21, and 24 h of vibration, the resulting solution was collected and stored at −20 °C with 10 ml fresh PBS re-added into the tubes. The effects of different environment pH on Schiff base breakage and liposome release was studied using the Phospholipid Assay Kit (Sigma-Aldrich, MAK122, USA). Similar to the above methods, the effects of different acidic environments on the release of NGF from microsol electrospun fiber scaffolds were also analyzed using the NGF ELISA Kit (R&D Systems, USA). The cumulative release curves were drawn, respectively.

MMP-3 and NGF protein concentrations/100 μL of cell culture supernatant were determined using a human MMP-3 ELISA kit (R&D Systems, Inc., Minneapolis MN, USA) and an NGF ELISA kit (R&D Systems).

To determine whether the TGF‐β inhibitor, SB431542, inhibits Ngf expression and NGF production in IVD cells, we examined the effect of SB431542 on TGF‐β-mediated Ngf expression and NGF production by IVD cells isolated from five mice. IVD cells were isolated using collagenase digestion, as described above. Disc cells were subsequently incubated in α‐minimal essential media (α‐MEM) with 10% fetal bovine serum in six‐well plates. One week later, IVD cells were stimulated with α‐MEM (vehicle), 10 ng/ml recombinant TGF‐β (Product no. 7666-MB, R&D Systems, Minneapolis, MN, USA), or 10 ng/ml recombinant TGF‐β + 1 µM SB431542 (Product no. S4317, Sigma Aldrich, St Louis, MO, USA) for 6 and 24 h. Thereafter, total mRNA was extracted and analyzed using qPCR. NGF concentration in the cell supernatant was determined using a commercial NGF ELISA kit (Product No. DY556, R&D Systems).