Detection of phosphatidylserine exposure on Mycoplasma hyorhinis by flow cytometry

Mycoplasma often contaminates eukaryotic cell cultures. Determining Mycoplasma cell viability is important when evaluating the cellular response to drug treatment. One method to test cell viability is using a membrane-impermeant nucleic acid dye that is generally excluded from viable cells. When cell death occurs, the plasma membrane phosphatidylserine (PS) is translocated from the inner side to the outer layer. This exposed PS has a high affinity for a Ca²⁺-dependent phospholipid-binding protein, Annexin V. In the present study we evaluated the potential application of Annexin V and propidium iodide (PI) in determining the cellular viability of Mycoplasma hyorhinis parasitized in host cell-culture supernatants. After Mycoplasma cells were treated with anti-Mycoplasma antibiotics, flow cytometry analysis was performed at a single-cell level and the untreated samples were included as negative controls. Subsequently, a robust association between PS exposure (Annexin V-positive) and drug dilution factor was observed. These data showed that Annexin V was an optimal probe for studying the physiological state of Mycoplasma in the early stages of death, characterized by disruption of phospholipid asymmetry.