Mplex microplate reader

ROS scavenging potential of Ful-containing cements and CMC/Gel were determined using a 1,1-Diphenyl-2-picrylhydrazine (DPPH) assay.^{[98 ,99 ]} DPPH solutions was prepared by dissolving DPPH (31.62 mg, Sigma-Aldrich, United States) in 80:20 v/v% solution of ethanol (400 mL, Thermo Fisher Scientific, United States) and deionized water. Respective cement samples (10 mg) were placed into DPPH solution (2 mL) and shook at 37 °C in the dark. At 1, 12, and 24 h, samples of DPPH (100 μL) were removed and absorbance was measured on a Tecan MPlex microplate reader at 517 nm. Absorbance values of DPPH solutions incubated with Ful containing cements ( $A_{\text{Ful}}$ ) were compared to absorbance readings of DPPH solution incubated with CMC/gel cement as shown in Equation (1) below.

MC3T3-E1 cells were seeded in a 12-well plate at 20 000 cells per well and incubated for 24 h to allow cells to adhere. After incubation, cells were treated with 1 mL of a 1:1 volumetric mix of osteoblastic induction medium and filtered extracts from respective cement formations (n = 3 wells per treatment). Cells were allowed to differentiate for 7 and 14 days, with media being replaced with fresh osteogenic media on days 4, 7, and 10. On days 7 and 14, cells were washed and lysed before extracting and purifying RNA using a PureLink RNA Mini Kit (Invitrogen, Waltham, MA), following the manufacturer’s protocol. Extracted RNA was quantified for quality and concentration using a NanoQuant plate on a Tecan MPlex microplate reader. cDNA was synthesized using an iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Hercules, CA). Quantitative real-time PCR (qRT-PCR) was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). Relative expression levels of runt-related transcription factor 2 (RUNX2) and Collagen Type 1 (COL1) were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the ΔΔCt method. Primer sequences are given in Table 2.