Murine fibroblast cell lines 3T3-A31 were cultured in culture medium containing DMEM (high glucose, D6429, Sigma-Aldrich, St. Louis, MO, USA), 10% FBS, and 1% penicillin/streptomycin. 70,000 cells/scaffold (with a diameter of 10 mm and a height of 4–5 mm) were seeded for a period of 14 days.
Metabolic activity was determined by the CellTiter 96® Aqueous One Solution Cell Proliferation (MTS) metabolic assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega corp., Madison, WI, USA), where the MTS tetrazolium compound was added directly to the cell culture medium in a 1:5 ratio. Metabolically active cells reduced the MTS reagent and generated a colored formazan dye that is soluble in cell culture medium. Formazan dye was quantified by measuring the absorbance at 490 nm, reference 690 nm using Tecan Infinite M200 Pro. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
The Quant-iT™ dsDNA Assay Reagent (Invitrogen) assay determined cell proliferation, as it quantified the amount of double-stranded DNA. The assay contains a fluorescent dye activated once it is bound to dsDNA. Fluorescence was measured at λex = 485 nm and λem = 523 nm. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
Cell viability was assessed by live-dead staining of three samples. 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF, Sigma-Aldrich, Saint Luis, MO, USA) was used to visualize the membranes of living cells and propidium iodide to visualize the nuclei of dead cells. Samples were observed using a Zeiss LSM 880 Airyscan confocal microscope. Excitation/emission was set as follows: BCECF λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm.
The cell distribution on the scaffold and the morphology were observed using 3,3'-Dihexyloxacarbocyanine Iodide (Invitrogen™) DiOC6(3)/propidium iodide (ThermoFisher Scientific™) staining. Excitation/emission was set as follows: DiOC6(3) λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm. The signal from the nuclei was further used to determine the depth of penetration of the cell into the scaffold.
The statistical significance was performed using one-way analysis of variance (ANOVA) in Sigma Stat software 3.5 (Systat Software, California, USA).
Metabolic activity was determined by the CellTiter 96® Aqueous One Solution Cell Proliferation (MTS) metabolic assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega corp., Madison, WI, USA), where the MTS tetrazolium compound was added directly to the cell culture medium in a 1:5 ratio. Metabolically active cells reduced the MTS reagent and generated a colored formazan dye that is soluble in cell culture medium. Formazan dye was quantified by measuring the absorbance at 490 nm, reference 690 nm using Tecan Infinite M200 Pro. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
The Quant-iT™ dsDNA Assay Reagent (Invitrogen) assay determined cell proliferation, as it quantified the amount of double-stranded DNA. The assay contains a fluorescent dye activated once it is bound to dsDNA. Fluorescence was measured at λex = 485 nm and λem = 523 nm. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
Cell viability was assessed by live-dead staining of three samples. 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF, Sigma-Aldrich, Saint Luis, MO, USA) was used to visualize the membranes of living cells and propidium iodide to visualize the nuclei of dead cells. Samples were observed using a Zeiss LSM 880 Airyscan confocal microscope. Excitation/emission was set as follows: BCECF λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm.
The cell distribution on the scaffold and the morphology were observed using 3,3'-Dihexyloxacarbocyanine Iodide (Invitrogen™) DiOC6(3)/propidium iodide (ThermoFisher Scientific™) staining. Excitation/emission was set as follows: DiOC6(3) λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm. The signal from the nuclei was further used to determine the depth of penetration of the cell into the scaffold.
The statistical significance was performed using one-way analysis of variance (ANOVA) in Sigma Stat software 3.5 (Systat Software, California, USA).
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