Cell clock cell cycle assay

Changes in the cell cycle dynamics of sEV-exposed B16F1 cells were analysed using the Cell-Clock cell cycle assay (Biocolor Ltd) according to the assay protocol. This assay can be used to distinguish the four major phases of the mammalian cell cycle using a vital redox dye, which is yellow, green or dark blue in G1, S/G2, and M phase cells, respectively. After staining, cells were photographed using an Axiovert S100 microscope (Zeiss) equipped by a Nikon D5000 camera. Images were analysed by the ImageJ software to determine the percentage of cells in each cell cycle phase. The experiment was performed with 4 repeats.

100 μl cell suspension of SKNAS and NB69 (1 × 10⁴ cells/well) was seeded in 96-well culture plates (Corning Incorporated). After culturing to 80% confluence the supernatant was removed and transfection media was added to the cells. 48 h post transfection, cells were counted using a 60 μm sensor for the Scepter handheld cell counter (Millipore) as per the manufactures instructions [29 ]. Cell proliferation was measured using the MTS/MPS Cell Titer 96® One solution Reagent (Promega) and detecting the color variation (FLUOstar Omega, BMG Labtech) as per the manufacturer’s recommendations. The absorbance values were normalized to the mock transfection and expressed as a percentage. All experiments were repeated three times.
Cell cycle analysis was performed using the Cell-clock cell cycle assay (Biocolor). Images were subsequently analyzed using Image J image analysis as per the manufacturer’s instructions. The data presented is the average of three biological replicates. Each experiment series was repeated in triplicate.

GC cell cycle analysis was performed using a Cell-Clock Cell Cycle Assay (Biocolor, Carrickfergus, UK) according to the manufacturer’s protocol.^{18 (link)} Pixel colour detection and counting of labelled cells were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). To test the reproducibility of the cell crock assay, we evaluated influences of ETNK2 KO on cell cycle regulation using the Muse Cell Cycle Kit (Merck Millipore, Billerica, MA, USA) under the manufacturer’s protocol.

100 µl cell suspension of SKNAS (1 × 10⁴ cells/well) was seeded in 96-well culture plates (Corning Incorporated). After culturing to 80% confluence the supernatant was removed and transfection media was added to the cells. 48 h post transfection, cells were counted using a 60 µm sensor for the Scepter handheld cell counter (Millipore) [26 ]. Cell proliferation was measured using the MTS/MPS Cell Titer 96^® One solution Reagent (Promega) and detecting the color variation (FLUOstar Omega, BMG Labtech) as per the manufacturer’s recommendations. The absorbance values were normalized to the mock transfection and expressed as a percentage. All experiments were repeated three times. Cell cycle analysis was performed using the Cell-clock cell cycle assay (Biocolor). Images were subsequently analyzed using Image J image analysis as per the manufacturer’s instructions. The data presented is the average of three biological replicates. Each experiment series was repeated in triplicate.

To evaluate proliferation and apoptosis, both CPC-P receiving EV-CM and control group were plated at a density of 2.5 × 10⁴ /cm² and stained with Cell-Clock Cell Cycle Assay and Cell-ApoPercentage Apoptosis Assay (both from Biocolor Ltd., Carrickfergus, United Kingdom), respectively, strictly following manufacturer’s instructions. In the first assay, cells were incubated at 37°C for 1 h with the redox dye supplied by the kit. Such dye is taken up by live cells and its uptake induces a distinct change in cell color, from light green to blue, specifically associated with G1- S- G2- or M-phase of the cell cycle. For ApoPercentage Apoptosis Assay, CPC-P receiving EV-CM or from control group, were first incubated with 3% hydrogen peroxide in the complete medium for 12 h, then stained with the ApoPercentage dye that is selectively imported by cells undergoing apoptosis. Microscopic analysis and quantification were performed by three independent observers with a Leica DM2000 LED microscope (Leica Microsystems) equipped with a digital camera Leica ICC50 HD (Leica Microsystems). Data were expressed as mean percentage of cycling cells over total cells ± SEM, for Cell-Clock Cell Cycle Assay, and as mean percentage of stained cells ± SEM over total cells for Cell-ApoPercentage Apoptosis Assay.