Bdnf elisa kit

Commercially available human DPSCs and human BM-MSCs (Catalogue # PT-5025 & PT-2501, respectively) were purchased from Pharma & Biotech (Lonza group Ltd, USA). C5aR antagonist—W54011 was acquired from Calbiochem (San Diego, CA, USA) and C5aR agonist from Anaspec (Fremont, CA, USA). MEM-alpha, PBS, fetal bovine serum, L-glutamine, and Antibiotic–Antimycotic were procured from Gibco Fisher Scientific (Waltham, MA, USA). Poly-D-Lysine coated (BioCoat, 12 mm) round German glass coverslips slips were purchased from Corning Fisher Scientific (Waltham, MA, USA). RIPA buffer was from Cell Signaling Technology (Danvers, MA, USA) and BDNF ELISA kit from R&D System (Minneapolis, MN, USA). Various antibodies were procured: anti-C5a receptor from Proteintech (ST. Louis, MO, USA), rabbit anti-BDNF from NovusBio (Centennial, CO, USA), mouse anti-NGF from BioLegends (San Diego, CA, USA), mouse anti-STRO-1, and mouse anti-pp38 from Santa Cruz (Dallas, Texas, USA).

The cells were seeded onto culture flasks to a final density of 1 × 10⁶ cells. After 24 h of culture, the cells were exposed to tunicamycin (0.5 µg/mL) with or without R- and S-ketamine (10 µM) for the next 24 h. After treatments, culture medium was collected and centrifuged at 250× g, 5 min. BDNF protein levels in the cell-conditioned media were determined using a BDNF ELISA kit, according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Data are represented as pg/mL protein.

The levels of inflammatory cytokines, such as IL-1α, IL-1β, IL-6 and TNF-α, in the cerebrospinal fluid were measured using commercial ELISA kits (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The expression levels of BDNF were measured using a BDNF ELISA kit (R&D system), according to the manufacturer’s instructions.

Supernatants or cell lysates from DPSCs culture, incubated with various above-mentioned treatments, were collected from cultures (cell lysates were collected using RIPA buffer) after 24 or 48 h and assayed using BDNF ELISA kit according to manufacturer’s protocol (R&D Systems). Briefly, a standard curve was constructed using standards and test samples in duplicate at increasing concentrations and values were normalized accordingly.

We evaluated the impact of paclitaxel and memantine on the hippocampal BDNF expression and the associated effects on hippocampal neurogenesis. Four groups with 2 different memantine treatment regimens were assessed: control, paclitaxel, memantine, and memantine + paclitaxel. In the pretreatment regimen (Experiment 2), the BDNF was assessed at 2 time points: 6 h after the last memantine dose and on Day 7 after completing the paclitaxel treatment. In the cotreatment regimen (Experiment 3), the BDNF was assessed on Day 7 after completing the paclitaxel and memantine treatment and Day 14. The mice were sacrificed by decapitation, and the bilateral hippocampi were removed. The hippocampal levels of the BDNF were measured by ELISA. The tissues were homogenized in lysis buffer with a protease inhibitor cocktail. Then, the samples were centrifuged at 9000× g for 15 min at 4 °C. The supernatant was then collected, and the BDNF was assessed with a BDNF ELISA kit (R&D Systems, Minneapolis, MN, USA). The data were calculated from the standard curves created with human BDNF for each plate and were expressed as the mean BDNF value (pg) per mg of total protein, which was used to normalize the BDNF results. The results were expressed as concentrations (pg/mL).

Supernatants or cell lysates from DPSCs and BM-MSCs cultures, incubated with various above-mentioned treatments, were collected from cultures after 48 h and assayed using BDNF ELISA kit according to manufacturer’s protocol (R&D Systems). Briefly, a standard curve was constructed using standards and test samples in duplicate at increasing concentrations and values were normalized accordingly.