2.3.2. SRB (Sulforhodamine B) Cell Proliferation Assay

The human keratinocyte HaCaT cell line (nontumorigenic, spontaneously immortalized cells) was purchased from Cell Lines Service (Eppelheim, Germany). Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) containing 10% fetal bovine serum (PAN Biotech), 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 mM HEPES (pH 7.3; Sigma-Aldrich, Chemie GmbH, Steinheim, Germany). Cell cultures were incubated at 37 ◦C in a humidified atmosphere with 5% CO2. The synthesized (co)oligoesters (samples 1–2) and unconjugated p-anisic acid were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions. Working solutions were made by combining stock solutions with a complete culture medium, and sterile filtered. The final concentration range of the studied compounds was 1–100 μg/mL. DMSO concentration in every culture medium (including control) was adjusted to 0.2%. As a positive control, treatment with 5% DMSO in the culture medium was applied.

In Vitro Toxicology Assay Kit, Sulforhodamine B Based (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) was utilized to study cell proliferation. Keratinocytes were seeded into 96-well plates at an initial density of 3 <sup>×</sup> 10<sup>3</sup> cells/well in 200 <sup>μ</sup>L of medium. Cells were allowed to adhere and grow for 24 h. Then, the medium was replaced with working solutions, and keratinocytes were cultured for the next 72 h. After treatment, the culture medium was aspirated, and keratinocytes were fixed with 10% trichloroacetic acid, washed with deionized water, and stained with 0.4% SRB (sulforhodamine B) dissolved in 1% acetic acid. The unincorporated stain was washed out with 1% acetic acid and the incorporated sulforhodamine B was solubilized in 200 μL of 10 mM tris(hydroxymethyl)aminomethane solution. Absorbance was measured at 570 and 690 nm (reference wavelength) using the MRX Revelation plate reader (Dynex Technologies, Chantilly, VA, USA).
