Universal microplate reader

Cell proliferation was determined using WST-8 dye (Beyotime Inst Biotech, China) according to our previous report [27 (link)]. Briefly, HSCs were treated with PDGF (10 ng/ml) for 24 h along with or without PQQ (0.3 μg/ml, 3 μg/ml and 30 μg/ml) or NAC (800 μg/ml), and then incubated with WST-8 dye. Absorbance was determined at 450 nm using a Universal Microplate Reader (Bio-Tek Instrument Inc.). All assays were performed in triplicate.

Proliferation analyses were performed to determine the cell vitality using MTT assays (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) (USB Corporation, Cleveland, OH, USA). The hPDLSCs were collected from three different donors. Each well (24-well plate) was seeded with 50,000 cells. The hPDLSCs were cultured on N. benthamiana-produced hDMP1 (2 µg/mL), commercial hDMP1 (2 µg/mL) (R&D Systems, USA), and WT (1 µg/mL) for 24 and 72 h. Then, as much as 500 μL of MTT reagent with a concentration of 0.5 mg/mL was added to every well and incubated at 37 °C for 30 min. Elution with 500 µL of glycine buffer (pH 10, 100 mM NaCl and 100 mM glycine: DMSO (1:9)) was performed. The absorbance was measured using a spectrophotometer at a wavelength of 570 nm on a universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). All experiments were performed in triplicate. Statistical analysis was performed using GraphPad 7 and one-way ANOVA.

SCAP in the control group and the E2 group were seeded into 96-well plates (Nunc, USA) at a density of 2 × 10³ cells/well or 24-well plates (Nunc, USA) at a density of 1 × 10⁴ cells/well and cultured in routine media or mineralization-inducing media (MM) containing α-MEM, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM ascorbic acid, 2 mM 2-glycerophosphate and 10 nM dexamethasone. Alkaline phosphatase (ALP) activity assay was performed as previously reported
[20 (link)] by using an ALP activity kit (Sigma-Aldrich) and normalized to total protein content in the cells at days 5 and 7. At day 14, alizarin red staining was carried out as described before
[21 (link)] and images were acquired using a scanner. Then, nodule staining was destained by 10% cetylpyridinium chloride (CPC) in 10 mM sodium phosphate for 30 minutes at room temperature. The calcium concentration was determined by measuring the absorbance at 526 nm with a universal microplate reader (BioTek Instruments). This experiment was performed in triplicate and the results are presented as the mean ± SD.

Cytotoxic and cell apoptosis assays were performed to evaluate the cell killing effects of P_HSP-TK/GCV on breast cancer cells. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer's protocol. Briefly, CCK-8 solution (100 μl) was added into the cell chamber and incubated for 2 h, and the absorbance of each chamber was measured at 450 nm using a Universal Microplate Reader (BIO-TEK Instruments, Minneapolis, MN, USA). Then, cell apoptosis assay was performed as described in the Annexin V-APC apoptosis detection kit using flow cytometry analysis (Becton Dickinson FACScan, Mount View, VA, USA). Then, the apoptosis index was analyzed by counting the number of Annexin V positive cells (red staining) per HPF (high-power field, 40×) in 10 slides for each group.