I control 1.9 magellan software

To determine the concentration of NGF, IVD cells were cultured in monolayer (250,000 cells/sample) and then lysed using 300 μL of Cell Lysis buffer (RayBiotech, Norcoss, GA, USA). Cell lysates were incubated for 48 h at room temperature and protein concentrations were determined using ELISA kits, according to the manufacturer’s instructions (RayBiotech, Norcoss, GA, USA). Cell culture media from degenerate IVD cells cultured in monolayer and in pellets was used to assess the concentrations of IL-6, IL-8, IL-1β, and TNF-α. One hundred and fifty microliters of monolayer culture media and pellet pre-treated and pooled post-treated media was used. ELISAs were performed as per the manufacturer’s instructions (RayBiotech, Norcoss, GA, USA). Colorimetric absorbance was measured with a Tecan Infinite M200 PRO (Tecan, Männedorf, Switzerland) spectrophotometer and analyzed with i-control 1.9 Magellan software (Tecan, Männedorf, Switzerland). Protein levels of the treated conditions and controls were then compared.

Protein concentrations of inflammatory cytokines IL-6 and IL-8 present in the collected culture media were measured using ELISA kits (RayBiotech, Peachtree Corners, GA, USA) according to the manufacturer’s instructions. Post-treatment CM of hMSC or DC pellets were pooled. Absorbance was measured using a TECAN Infinite M200 PRO plate reader with i-control 1.9 Magellan software (TECAN, Männedorf, Switzerland). Protein levels of untreated and o-Vanillin-treated samples were compared.

Sulfated glycosaminoglycans (GAGs) released in the CM of hMSC and DC pellets was quantified using DMMB assays as previously described [17 (link)]. CM of hMSC or DC pellets was pooled from the respective cell type and treatment. A serial dilution of chondroitin sulfate (C9819, Sigma-Aldrich, Oakville, ON, Canada) was used to generate the standard curve. DMMB dye was added to samples followed by an immediate measurement of the absorbance at 405 nm using a TECAN Infinite M200 PRO plate reader with i-control 1.9 Magellan software (TECAN, Männedorf, Switzerland). Data of co-culture groups were normalized with the formular (Normalized GAG concentration = GAG concentration/DC percentage).