Fitc phalloidin

The effect of the films on cell proliferation was performed with L929 fibroblasts using the Cell Counting KIT-8 (CCK-8). The sterilized samples (6 mm diameter) were placed in 24-well plates and immersed in a culture medium (Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum) until cell seeding. L929 cells were seeded at a density of 1×10⁶ cells per well and incubated for up to 6 days at 37°C under 5% CO₂. At two time points (3 and 6 days), the medium was replaced by a new medium containing 10% CCK-8, and the cells were cultured for 2 h. After being transferred into a 96-well plate, the resulting medium of 100 μL was measured with a microplate reader at 450 nm. The relative growth rate (RGR) was calculated as A/A₀ × 100%, where A and A₀ are the resulting absorbances with and without adding to the film samples, respectively.
For observation of cell morphology, cells were spread on a confocal dish containing the film samples and cultured under the same conditions. After 6 days of incubation, the medium containing the films was removed, and the cells were fixed in 4% paraformaldehyde. Before observation by confocal laser scanning microscopy (CLSM; Nikon A1R, Japan), the cells were dark-stained with FITC-phalloidin (Cytoskeleton Inc., America) for 40 min and DAPI (Beyotime Institute of Biotechnology, China) for 10 min, sequentially.

After 24 h of exposure to the half of the IC₅₀ and combined IC₅₀ treatments, the morphological changes induced by the treatments were assessed using the triple staining protocol previously published by our research team [23 (link)]. The cells were incubated on a chamberslide for 24 h with the treatment added. After 24 h, the cells were washed and stained with Mitotracker (Thermo Fisher Scientific, Waltham, MS, USA) for labeling the mitochondria (Texas Red), Phalloidin-FITC (Cytoskeleton, Denver, CO, USA) for labeling the actin filaments, and DAPI (Thermo Fisher Scientific, Waltham, MS, USA) for labeling the nucleus. Cells were analyzed under an Olympus FLUOVIEW FV1200 laser scanning fluorescence confocal microscope (Olympus, Tokyo, Japan).

To investigate the morphological changes induced by the 48 h treatment with L. alpinum extract (400 µg/mL), cells were analyzed in bright field with an inverted microscope. The same instrument was used in combination with a fluorescent source (HBO50, same manufacturer) for details regarding the cytoskeleton and nucleus, after staining these cell compartments with Phalloidin-FITC (for the cytoskeleton) (Cytoskeleton, Denver, CO, USA) and DAPI (for the nucleus) (Thermo Fisher Scientific, Waltham, MS, USA) using a previously published protocol [50 (link)].

The fluorescent staining protocol used DAPI (Abcam, Cambridge, UK) for the labelling of the nucleus and Phalloidin-FITC (Cytoskeleton Inc., Denver, CO, USA) for the cytoskeleton. After treatment, the cells were fixed in 4% paraformaldehyde followed by 0.5% Triton × permeabilization for 1 h. The cells were incubated at 37 °C and 5% CO_2. Thereafter, 100 µL of 200 nM Phalloidin-FITC was added and the samples were incubated at room temperature under no illumination for 30 min. 200 µL of 100 nM DAPI was added over the coverslip for 30 s and washed with a phosphate saline buffer. The coverslips were mounted with 90% glycerol. Images were captured using a UPLSAPO40 × 2, NA:0.95 objective (Olympus, Tokyo, Japan) and excitation wavelengths/emission windows were automatically selected according to the fluorescence dye spectral information database inside the acquisition software (FW10-ASW, Olympus, Tokyo, Japan).