Eradication of Mycoplasma contamination in HeLa cells using neomycin resistance gene introduction and aminoglycoside G418 (Geneticin) treatment.

Mycoplasma contamination remains a persistent problem in continuous cell culture, compromising cellular physiology, altering gene expression, and potentially leading to erroneous experimental conclusions. Mycoplasmas can profoundly affect cultured cells, underscoring the need for efficient eradication strategies. Here, we developed a simple and effective method to eliminate Mycoplasma from HeLa cell lines. A neomycin resistance gene was introduced into Mycoplasma-contaminated cells, conferring resistance to aminoglycoside-induced cytotoxicity. Subsequently, a high concentration of the neomycin analogue G418 (Geneticin), combined with single-cell cloning, was applied to achieve complete removal of the Mycoplasma contamination. Mycoplasma presence and clearance were confirmed by PCR targeting the 16 S rRNA gene and immunofluorescence using a Mycoplasma-specific monoclonal antibody. This genetic-antibiotic combination proved technically simple and highly effective for long-term Mycoplasma eradication in continuous cell lines. To investigate the functional impact of Mycoplasma contamination, we compared protein expression following transient transfection with GFP and FAM-labeled reporters in Mycoplasma-eradicated versus Mycoplasma-contaminated cells. Fluorescence analysis revealed a marked increase in transfection efficiency and reporter expression in Mycoplasma-eradicated cells. Our findings provide an effective strategy for eliminating Mycoplasma contamination in cell line cultures, ensuring the reliability of cell-based research and the accuracy of experimental data.