Fitc and texas red filters

ADSCs were seeded with a density of 1000 cells per 1 cm². In 24 h the control growth medium was changed for the experimental one. For the cytotoxicity assessment the cells were stained with РI (propidium iodide) (Sigma-Aldrich, USA) and FDA (fluorescein diacetate) (Sigma-Aldrich, USA) after 24 h and 72 h in culture. PI does not penetrate into living cells; therefore it stains only those in which the integrity of the cell membrane was broken. FDA is not a fluorescent molecule, but when penetrated into living cells is converted into a fluorescent form, fluorescein. The number of dead and living ADSCs in different groups was counted using fluorescence microscopy (FITC and Texas Red filters; Carl Zeiss, Germany).
Cytotoxicity was calculated as the ratio of living cells to the total number of cells and was expressed as a percentage according to the formula:
Cytotoxicity= (total number of cells / number of living cells)×100%.

The experiments with the use of human cell culture in vitro were carried out following the human experiment issues of the Code of Ethics of the World Medical Association (Declaration of Helsinki, 19 October 2013). In all cases, donors of ADSCs were the informed voluntary. Adipose-derived Stem Cells (ADSCs) were isolated from the lipoaspirate by enzymatic digestion in 0.1% collagenase IA and 0.1% pronase with 2% fetal bovine serum (FBS). This cell suspension was then transferred to 25 cm² cell culture flask (SPL, Gyeonggi-do, Korea) and cultured in the following control growth medium: modified MEM-α with 10% FBS, 2.0 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 1.0 ng/mL bFGF-2. (All media and reagents were obtained from Sigma-Aldrich, St. Louis, MO, USA.) The cells were cultured in multi-gas incubator CB210 (Binder, Tuttlingen, Germany) at +37 °C under saturated humidity, 5% CO₂ and 5% O₂. For the cytotoxicity assessment, the cells were seeded on scaffolds at a density of 2 × 10⁵ cells per 1.0 cm³ of materials. After 48 h of incubation, samples were stained with PI (propidium iodide) and FDA (fluorescein diacetate). The number of dead and living ADSCs in different groups was counted, using fluorescence microscopy (FITC and Texas Red filters; Carl Zeiss, Jena, Germany) and ZEN 2012 software [22 (link)].

Standard protocol was used for the FDA and PI assay [29 (link)]. The cells were stained with РI (propidium iodide) (Sigma-Aldrich, USA) and FDA (fluorescein diacetate) (Sigma-Aldrich, USA) after 24 h and 72 h in culture. PI does not penetrate into living cells; therefore, it stains only those in which the integrity of the cell membrane is broken. FDA is not a fluorescent molecule, but when penetrated into living cells, it is converted into a fluorescent form, fluorescein. The number of dead and living cells in different groups was counted using fluorescence microscopy (FITC and Texas Red filters; Carl Zeiss, Germany).
Viability was calculated as the ratio of living cells to the total number of cells and was expressed as a percentage according to the formula: $\begin{array}{c}\text{cell\hspace{0.17em}viability}=\left(\frac{\text{number\hspace{0.17em}of\hspace{0.17em}living\hspace{0.17em}cells}}{\text{total\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}cells}}\right)\times 100\%.\end{array}$