Mtt assay kit

The siRNAs were delivered to the grown cells by using reverse transfection with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions. The cells were procured from ATCC^® and further grown in a standard culture plate at defined conditions in DMEM. The standard siRNA dilutions were prepared at various concentrations (50, 25, 10, and 5 nM) from 50 μM stock solutions by adding 100 μL of Opti-MEM and Lipofectamine 2000 and further incubated at room temperature for 30 min. The complex mixture was delivered slowly to the grown cells, which were allowed to grow further for 24 h at 37 °C. The cellular toxicity of the siRNAs in different cell lines was analyzed by using an MTT assay kit (Invitrogen) following the manufacturer’s instructions. The absorbance was measured at 570 nm using a SpectraMax i3x imaging cytometer, and the standard formula was applied for the cytotoxicity calculation. The detailed protocol for the cytotoxicity assay has been described previously [29 (link)].

Cell viability was explored with the MTT assay kit (Invitrogen). In short, transfected DU145 or LNCaP cells were seeded at the density of 1 × 10³ cells per well to 96-well plates, washed in phosphate-buffered saline (PBS; Sigma-Aldrich) and sequentially incubated for 4 h using MTT solution. Upon incubation, medium was exchanged into DMSO and cells were incubated for 10 min. OD490 nm value was read with a microplate reader. Assay was implemented in at least triplicate.

Osteoblast cells (MC-3T3) were cultured in Dulbecco's Modified
Eagle's Medium supplemented with fetal bovine serum (10% v/v); cells were
incubated at 37 °C in a humidified atmosphere with 5%
CO₂. Cells were grown till confluence was reached, washed
twice with sterile PBS and detached with trypsin.
Samples were placed in 24-well plate with 500 μl of osteoblast cell suspension and incubated with the composite gel at
37 °C in a humidified atmosphere with 5%
CO₂. After 2 days, the medium was removed and
1 ml fresh medium without red phenol was added. Osteoblast
cell viability was assessed using the MTT assay kit (Invitrogen, Paisley, UK) with
20 μl of the reagent solution, prepared according to the
manufacturer instructions, added to each well. After incubation for 2 h at 37 °C in a humidified atmosphere with 5%
CO₂ all the solutions were removed and the MTT solubilization
solution was added. When full dissolution of the crystals occurred, 100 μl of liquid was transferred to a 96-well plate where the absorbance
of each sample was read at 570 nm.

The viabilities of H9c2 CMs after different treatments were determined using the MTT Assay Kit (Invitrogen, NY) by following the manufacture’s protocol. The CMs culture was replaced with 100 µl of fresh culture medium. Cells in 96-well plate were added in 10 µl of 12 mM MTT solution and incubated at 37°C for 4 h. Then 100 µl of the sodium dodecyl sulfate (SDS)-HCl solution was added to each well and incubated at 37°C for 4 h. Finally, the 96-well plate was read by a microtiterplate reader (Packard) at 535 nm. The percentage of viability was defined as the relative absorbance of the treated cells versus the untreated controls [19] (link).

All
assays and fluorescent probes, including the MTT assay kit (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide), calcein-AM (a live cell marker), propidium iodide (PI, a
fluorescent DNA intercalating probe), and carboxy-dihydrodichlorofluoresceine
diacetate (carboxy-H₂DCFDA, a general oxidative stress
indicator), were purchased from Invitrogen (ThermoFisher Scientific,
U.S.A.). Trypan blue solution and Triton X-100 solutions were purchased
from Sigma-Aldrich (Poland).