Cell counting kit

The percentage cellular viability/mortality was initially assessed via staining with Trypan blue 0.4%. Cell proliferation was analyzed via the reduction in tetrazolium salt WST-8 using the Cell Counting Kit (CCK, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. A total of 1000 cells for HepG2 or 5000 for LX-2 were seeded per well in 96-well plates and cultured in the different conditions for 24, 48, and 72 h. An aliquot of 10 µL of CCK was added per well, and the wells were incubated for 4 h at 37 °C and 5% CO₂. The absorbance was read at 450 nm with a microplate reader. A seven-point standard curve (1000–24,000 cells) was used to interpolate the data and calculate the cell number.

Anti-HIV activity of RBV bearing polymers was conducted using TZM-bl cell line as reported earlier.^{17 (link)} This cell line is engineered with a luciferase/β-galactosidase reporter system under control of an HIV long terminal repeat promoter, and expresses the CD4 receptor and CCR5/CXCR4 co-receptors which are indispensable for HIV to infect the cell. Briefly, TZM-bl cells were preincubated with polymers at conc. 0.1 g L^–1 for 24 h, following which cells were infected with HIV-1 Bal strain (14 TCID50). After 48 h level of infection was quantified via luciferase assay (Perkin-Elmer). Cell viability was measured using Cell Counting-Kit (Sigma-Aldrich).

For Cell Counting Kit (CCK) assay, a Cell Counting Kit reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to the well and incubated for 2 h. Absorbance value was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). For BrdU incorporation, BrdU labeling reagent (Roche Diagnostics, Indianapolis, IN, USA) was added to each well and cells were reincubated for 24 h at 37°C. After removing CCM, cells were fixed and DNA denatured by adding 200 mL of FixDenat (Roche Diagnostics, Indianapolis, IN, USA) for 30 min at room temperature. After removing FixDenat, anti-BrdU-POD working solution was added to each well and left at room temperature on a shaker for 90 min. Each well's absorbance was measured at 370 nm with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

7000 cells were seeded in 96 well plates and incubated for 24 hours at 37 °C in a humidified incubator with 5% CO₂. The media was replaced, and cells were exposed to alisertib (350 nM for C10 and 35 nM for SW48) for 48 hours followed by 72 hours of cetuximab treatment (10 ng/µL for C10 and 1 ng/µL for SW48). The alisertib concentration was chosen to cause less than 30% of death after 72 hours of treatment. In the case of cetuximab, the minimum concentration that elicited an optimal response after combination with alisertib was selected.
To determine the survival percentage of treated cells relative to the control, Cell Counting Kit (CCK8, Sigma Aldrich #96992) was used according to the manufacturer’s instructions.

Cellular Viability was assessed using the Cell Counting Kit (CCK8, Sigma). Briefly, 5 μl of CCK8 solution was added in 50 μl PBS to the cells and incubated for 60 min at 37°C prior to measurement of absorbance at 450 nm in the Envision II.

Cell proliferation on HA coated surfaces was measured using the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt] assay with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). MC3T3-E1 cells were cultured in a non-osteogenic standard medium on untreated and plasma-treated HA-coated culture plates (BD BioCoat™ Osteologic™) at an initial density of 3 × 10³ cells per well. The non-osteogenic standard medium consisted of α Eagles’s minimal essential medium (αMEM, Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Lot No.002095, JRH Bioscience, Lenexa, KS, USA), and antibiotics (100 U/ml of penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml of amphotericin B; Sigma-Aldrich, St. Louis, MO, USA).
At 1, 2, and 4 day time points, 10 μL of the Cell Counting Kit solution was added into each well, followed by incubation of the culture plates at 37°C in 5% CO₂ for 2 hours. The absorbance was read at 450 nm with an automatic enzyme-linked immunosorbent assay reader (Multiskan Ascent MS353; Thermo Fisher Scientific).