Tetracolor one

Hela cells (1 × 10³) or HCP4 cells (2 × 10³) were seeded into 96-well plates for 24 h and then treated with Cisplatin or statins at the maximum concentration indicated and 2-fold serial dilutions. For combination treatment with Lovastatin and Cisplatin, the indicated fixed concentration ratios were used. After 72 h, the surviving cells were stained with cell proliferation assay kit (TetraColor ONE; Seikagaku Corporation, Tokyo, Japan) for 2–3 h at 37°C and absorbance was measured at 450 nm, according to the manufacturer’s protocol. To measure the half maximal inhibitory concentration (IC50) in each experiment, CalcuSyn software version 2.0 (Biosoft, Cambridge, UK) was used. For combination treatment of Cisplatin and Lovastatin, the combination index (CI) calculated by CalcuSyn software was employed as previously reported [42 (link)]. For analysis of transfectants, cells expressing GFP were counted using a LUNA-FL™ Dual Fluorescence Cell Counter (Logos Biosystems, Gyunggi-do, South Korea) according to the manufacturer’s protocol.

Cell growth was measured by an MTT assay. For the MTT assay, LNCaP and 22RV1, 3 × 10³ cells/well were seeded in 96-well plates. C21 was added daily with replenishment in 22RV1 and without it in LNCaP cells. After incubation, 10 µL TetraColorOne (Seikagakukogyo, Tokyo, Japan) was added to each well, and the absorbance was quantified according to the manufacturer’s protocol.

The procyclic form of the parasite (2 × 10⁵ cells per well) Trypanosoma congolense IL 3000 was cultured for 48 h in TVM-1 medium [27 (link)] in 96-well plates with various concentrations of aurantoside L (1). Ten microliters of TetraColor ONE (Seikagaku Biobusiness, Tokyo, Japan) was added to each well. After 4 h, the absorbance of the samples was read at 450 nm using a microplate reader.

A murine macrophage cell line, RAW264.7, was purchased from American Type Culture Collection (Manassas, VA) and was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). Liposomal clodronate (lipo-CL2MDP) was kindly gifted by Dr N. van Rooijen [11 (link), 12 (link)]. Liposomal PBS (lipo-PBS) was used as a control. RAW264.7 cells treated with the indicated concentrations of lipo-CL2MDP or lipo-PBS were seeded at a density of 10,000 cells/well in a 96-well plate and incubated for 48 hours at 37°C. After the indicated time, the viability of the cells was assessed by a colorimetric assay (TetraColor One®; Seikagaku Co., Tokyo, Japan) as described elsewhere [10 (link), 13 (link), 14 (link)]. Briefly, 10 μL of TetraColor One® was added to each well, and the mixture was incubated for an additional 4 hours to measure the viability of cells. Absorbance at 450 nm was monitored and the IC₅₀ values of the cells were calculated.

Cells were plated at a density of 2 × 10⁴ cells/ml and cultured in RPMI supplemented with 5% FBS overnight. After siRNA treatment, the cells were further cultured in RPMI containing N2 supplement and 20 ng/ml EGF for 48 hours. Viable cells were measured in triplicate using TetraColor One (Seikagaku) with reference to the viability of mock‐treated cells.

The proliferative effects of FGF2 on stromal cells (MS-5 and S-17), mouse osteoblasts (7F2), and human leukemia cells (NCO2 and Meg-A2) were assessed by a colorimetric assay (TetraColor One; Seikagaku Co., Tokyo, Japan) as described elsewhere48 (link). Briefly, cells were washed twice with PBS, suspended in culture medium (DMEM containing 10% FBS), plated (stromal cells: 1000 cells, osteoblast: 2000 cells, leukemia: 20000 cells/well in 0.2 mL culture medium) onto 96-well plates with the addition of FGF2 at various concentrations (0–100 ng/mL), and incubated for 72 hours. Subsequently, 10 μL of TetraColor One reagent was added to each well, and absorbance at 450 nm was measured eight hours later.