The APC was funded by Warsaw University or college of Existence Sciences. Institutional Review Table Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented with this study are available within the article. Conflicts of Interest The authors declare no conflict of interest. of the JC-1 reddish/green fluorescence intensity ratio revealed related levels of mitochondrial membrane potential in cells growing on graphene-coated and uncoated slides. These results indicate that graphene monolayer scaffold is definitely cytocompatible with connective cells cells examined and could be beneficial for cells executive therapy. ? 0.01) increased with graphene like a scaffold for cells, while shown in Number 6. Mitochondrial activity raised from 100 3.6% in control cells ITGA9 grown on glass substrate to 122 5.8% in cells seeded on graphene scaffold. In vitro studies of graphene substrate in direct contact method did not show any harmful effects on BALB/3T3 cells. It can be concluded that the use of graphene substrate enables the adhesion and enhance the mitochondrial activity of BALB/3T3 cells. 3.5. Mitochondrial Network Morphology The morphology of mitochondrial network brings important information concerning the cell health and its function. Consequently, to visualize mitochondrial morphology we used two dyesMito Tracker Green FM (in living cells) and Mito Red (in fixed cells). There were no changes in the mitochondrial network morphology and distribution in BALB/3T3 cells cultivated on graphene. Images of cells growing on both mediaglass and graphenewere characterized by an even distribution of the mitochondrial network and well connected (Number 7). Open in a separate window Open in a separate window Number 7 The morphology of mitochondrial network after staining with Mito Tracker Green FM and Mito Red Green fluorescence in living cells and reddish fluorescence in fixed cells. Magnification in squares presents different phenotypes of mitochondria: right rods, twisted rods, branched rods and loops. The mitochondrial network was neither too fragmented nor too Amylin (rat) elongated and does not show swollen and irregular structures or huge spherical mitochondria. Mitochondria were oriented parallel to the long axis of BALB/3T3 cells. Mitochondria of cells growing on control substrate and graphene can be described as networked and rod-like with different phenotypes: right rods, twisted rods, branched rods and loops (donut). 3.6. Mitochondria Membrane Potential Circulation cytometry evaluation of Amylin (rat) mitochondrial membrane potential using JC-1 dye clearly showed that graphene did not cause decrease of this parameter in BALB/3T3 cells. The percentage of cells with low (green color) and high (red color) membrane potential in the control and graphene organizations was comparable. More cells with reduced potential (blue color) were shown in the control group versus the group with graphene coated slides (Number 8). Open in a separate window Number 8 Evaluation of mitochondrial membrane potential using JC-1 dye. Cytograms of JC-1-stained cells; Red populationscells with high membrane potential. Green populationscells with low membrane potential. Blue populationscells with high mitochondrial depolarization. Pub chart of reddish/green fluorescence intensity percentage of JC-1-stained cells. Data from three self-employed experiments are offered as mean SD (standard deviation) (n = 10,000 cells). ** – statistically significant variations. Nearly 100% of H2O2-treated cells showed a definite and significant decrease in the mitochondrial membrane potential (blue color). Additionally, cells exposed to H2O2 showed hyperfragmentation of the mitochondrial network, as determined by fluorescence microscopy (Number 9). Open in a separate window Number 9 Fluorescence microscopy showing mitochondrial network in cells growing on graphene and glass substrate. Staining cells with JC-1 shown the influence of hydrogen peroxide on mitochondria. There were no longer string formed mitochondria-like in control group and graphene substrate, instead punctate and inflamed mitochondria occurred. In the mean time, between cells cultured on graphene-coated and uncoated slides there were no visible changes in the level of green or reddish fluorescence of JC-1. 4. Conversation Graphene offers potential to be used in medical fields Amylin (rat) and composite enhancement, amongst additional uses. Biosafety of nanomaterials offers caused increased attention from scientists who are investigating their effects within the cells, animals and environment [23,24,25]. Comparative studies on graphene cytotoxicity help to efficiently apply these materials in medical fields. That is why the main goal of this Amylin (rat) study was to determine the cytotoxicity of graphene by in vitro checks on murine BALB/3T3 fibroblast. The research provides additional data within the suitability of graphene monolayer for being used like a scaffold for cells in regenerative medicine. Previously, we checked biocompatibility of pristine graphene with L929 fibroblast cells ..