T4049

HeLa, HeLa S3 and Phoenix A cell lines (originally from ATCC) were grown in Dulbecco’s modified eagle medium (D5796, Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (F7524, Sigma Aldrich). Cells were incubated at 37°C and 5% CO₂ and passaged using Trypsin-EDTA (0.25%, T4049, Sigma Aldrich).

Mice were humanely euthanized and rinsed in 70% ethanol. The liver tumor (2–3 g) was dissected, washed in PBS, and minced into ~1 mm fragments using a scalpel blade. All procedures were operated in the hood using autoclaved dissecting tools. The tumor fragments were digested with 0.6 mg/L collagenase (Sigma-Aldrich, C5138) at 37 °C for 15–30 min, then filtered through 100 μm nylon mesh cell strainer (BD, 352360) and spun (700–1000 rpm, 5 min). The cell pellet was resuspended and cultured in 5 ml DMEM with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin at 37 °C and 5 % CO₂ for ~4 weeks. The primary liver tumor cells were generated. To obtain a stably passaged cell line, 1 × 10⁷ primary tumor cells were injected into flanks of 6–8 week-old FVB/N mice. When the subcutaneous tumor developed, tumor tissue was dissected into small pieces and digested with trypsin (Sigma-Aldrich, T4049). The new generation of liver tumor cells (P0) were cultured and passaged in vitro for 3 times. Then 1 × 10⁷ “P0” cells were implanted into the flanks of a new recipient FVB/N mouse. These procedures were repeated for three rounds. The stably passaged cell line from c-MET/sgp53 HCC (MP cell) was finally generated.

The MTT (methyl-thiazol-tetrazolium) assay was used to test cellular viability
towards three different types of muscle cells. Briefly, cells in the culture
flasks were harvested using 0.25% trypsin-EDTA solution (Sigma^®,
T4049). C2C12 (1.5 × 10⁶ cells/well), A7r5 (5 × 10⁴cells/well) and H9c2 (1.5 × 10⁴ cells/well) were incubated at 37°C in
5% CO₂ for 24 hours. The cells were treated with β-CTX-2 fraction
(0.001-0.8 mg/mL; n = 3). Each experiment was carried out in triplicate. Changes
in cell morphology of cells were identified using light microscopy with
magnification 100x following 0 and 24 hours of incubation in the presence of
toxin. Next, following 24 hours of culture, cells were exposed to MTT by the
additions of 12 µL 0.5% MTT solution. After 4 hours, 100 µL dimethyl sulfoxide
(DMSO) was added and Formazan color was detected at 570 nm using a plate reader
(Beckman Coulter^TM, AD340). Cells treated with sterile PBS were used
as a negative control.

Cell viability was measured by trypan blue
(9 (link)). Trypsin/Ethylenediaminetetraacetic acid
(EDTA, T4049, Sigma, USA) cell suspensions
of the culture ASCs were used to measure cell
viability. A total of 20 μl of the cell suspension
(500 cells/μl) and 20 μl of trypan blue reagent
(T6146, Sigma, USA) were mixed and incubated
at room temperature for 5 minutes. Viable
cells were counted using a Neubauer hemocytometer
under a light microscope (Olympus
IX71). MTT assays were also used to determine
the effect of ZnO-NPs on cell viability and proliferation.
Briefly, cells were maintained with
culture media for 24, 48 and 72 hours in 24-
well plates. MTT (M2128, Sigma, USA) was
then added to each well (0.5 mg/ml) and cells
were further incubated for 4 hours at 37˚C. We
removed the supernatants and 700 μl of dimethyl
sulfoxide (DMSO, D2650, Sigma, USA)
was added to each well to dissolve the formazan
product. A 680 Microplate reader (BioRad, Hercules,
CA) was used to measure absorbance at
540 nm. MTT assay values were expressed as
the percentage of corresponding average values
in control cultures. Absorbance is in proportion to
the number of living cells in a sample. Thus, the
MTT assay indicates the extent of cell proliferation
(17 (link)).

HELA cell (CCL-2™, ATCC, USA) was chosen as the object for its easy-culture, uniformly growth and fast proliferation. Accordingly the cell culture medium is made of 89 vol% DMEM (Dulbecco’s Modified Eagle’s Medium, D5796-Sigma Aldrich, Switzerland), 10 vol% FBS (Fetal Bovine Serum, F6765- Sigma Aldrich, Switzerland) and 1 vol% P/S (Penicillin-Streptomycin, P4333-Sigma Aldrich, Switzerland). The cells were grown at 5% CO₂, 100% humidity and 37 °C for 5 days untill cells covered over 90% of the flask (3151-Corning, USA). The culture medium was replaced with 4 mL of PBS (Phosphate buffered saline, P5368- Sigma Aldrich, Switzerland) to wash away the dead cells. After dispersing the PBS, 2 mL of trypsin (T4049- Sigma Aldrich) was added to the flask for 3 min to detach the cells. The cells were transferred to a 15 mL tube filled with 8 mL of culture medium and centrifuged. After removing the medium from the tube, fresh medium was added to resuspend cells to a concentration of 10³ cells/mL.