Cx31 p

The TUNEL kit (Thermo Fisher) was performed to identify apoptosis in exosome-treated/untreated HCC cells. After the prescribed treatment according to the manufacturer’s protocol, Huh7 or HA22T cells were fixed by incubation with 4% (w/v) paraformaldehyde for 15 min at 4 °C. Next, the desired cells were sequentially incubated with TUNEL reaction liquid and DAPI for staining. Where DAPI was utilized to stain the nucleus (blue), and TUNEL staining (green) was applied to label apoptotic cells. Finally, fluorescence microscopy (CX31-P, Olympus) was employed to determine the number of TUNEL-positive cells after magnified imaging, and the degree of apoptosis was evaluated.

The ionic liquid [EMI][TFSI] was initially dried in vacuum for 24 h at a temperature of 70 °C. Next, P(VDF-HFP) and [EMI][TFSI] were co-dissolved in acetone with a weight ratio of 1:4:7. The ion gels were further dried in vacuum at 70 °C for 24 h to remove the residual solvent, after which it was cut with a razor blade, and then laminated onto the substrate of choice. To determine the nature of interaction between the ionic liquid and polymer matrix, FTIR spectroscopy was performed using FTIR spectrum GX, PerkinElmer. The plasticizing effect was investigated using DSC (DSC TA Instruments 2010) at a ramping rate of 10 °C min⁻¹. The samples were tightly sealed in aluminium pans, and the measurements were carried out while heating up the sample to 200 °C, followed by cooling down to –80 °C, at a heating and cooling rate of 10 °C min⁻¹. The degradation (working) temperature of the ion gel was measured by TGA (TGA-Q500). The self-healing nature of the ion gels was observed and captured under a polarizing optical microscope (Olympus, CX31-P).

A sterile cylindrical diamond bur (Teezkavan, Iran) was used for inciso-apical sectioning of mesial and distal surfaces. The specimens were immersed in 2.5% glutaraldehyde at 4°C for 24 hr and were then dehydrated by using 70%, 85%, 90%, and 95% ethanol once and 100% ethanol twice, each for 20 min. They were then dried at room temperature. The sections were gold sputter-coated and underwent SEM assessment (CX31P, Olympus, Tokyo, Japan) to ensure biofilm formation.