Brdu cell proliferation elisa kit

Cell viability: shNTC or shFMR1 transduced GSCs were plated at density of 2 × 10³/ml in 96 well plates in triplicate. Cell viability was monitored by counting the cells and confirmed by using the CellTiter-Blue™ Viability Assay (Promega). Cell proliferation: was evaluated by Bromo-2′-deoxyuridine (BrdU) incorporation using BrdU Cell proliferation ELISA kit (Abcam, Cod. ab126556) following the manufacturer's instructions. The motility of transduced GSCs was evaluated by plating in Corning FluoroBlok^TM Multiwell Inserts System (Corning Life Sciences, Tewksbury, MA), according to the manufacturer's instruction. Briefly, 3x10³ cells were added to the upper chambers in stem cell medium without growth factors (GFs). GF completed medium was used as chemoattractant in the lower chambers. The plates were incubated for 48 h at 37 °C, after which the fluorescent dye calcein acetoxymethylester (calcein AM, Life Technologies) was added to the lower chamber for 30 min. The cell viability indicator calcein AM is a non-fluorescent, cell-permeant compound that is hydrolyzed by intracellular esterases into the fluorescent anion calcein and can be used to fluorescently label viable cells before microscope observation. The number of migrated cells was evaluated by counting the cells after imaging acquisition using a fluorescence microscope (Nikon Eclipse TS100).

The effects of astaxanthin on the DNA synthesis and proliferation were assessed using the 5-bromo-2-deoxyuridine (BrdU) Cell Proliferation ELISA Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, after 24 h treatment of EqASC cells with astaxanthin at concentrations of 10 and 20 μg/mL, BrdU reagent was added to all culture media and incubated at 37 °C during 72 h. After fixation of the cell, the detection of the incorporated BrdU was performed using anti-BrdU primary monoclonal antibody and secondary goat anti-mouse IgG conjugated with horseradish peroxidase (HRP). HRP substrate degradation was measured using a plate reader spectrophotometer (Spectrostar Nano; BMG Labtech, Ortenberg, Germany) at a 450 nm wavelength.

For ex vivo proliferation, splenocytes were isolated 21 days p.i. from vehicle- and silybin-treated mice. To test the proliferation efficiency, the cells were treated with or without stimuli [25 μg/ml of MOG₃₅₋₅₅ or 5 μg/ml of concanavalin A (Con A)]. Cell proliferation was determined by the BrdU-incorporation test using BrdU Cell Proliferation ELISA Kit (Abcam, cat no. ab126556).

Splenocytes were isolated from ddH2O-treated and FSD-C10-treated EAE mice on day 12 p.i., and cultured with or without 25 μg/ml MOG_35–55 peptide for 3 days. Cell proliferation was determined by BrdU-incorporation test using BrdU Cell Proliferation ELISA Kit (Abcam). Supernatants from splenocytes were collected at 72 h to measure concentration of IFN-γ, IL-17, TNF-α, and IL-10 using ELISA kits (R&D System, Minneapolis, MN).