The human Ca Ski (HVP16, 400 to 600 copies per cell) and SiHa (HVP16, 1 to 2 copies per cell) cell lines, the HeLa (HPV18, 20 to 40 copies per cell) cell line and the HPV negative C33-A cell line were obtained from ATCC (Manassas, VA, USA) and routinely tested for the presence of HPV in our laboratory. Ca Ski and SiHa cells were maintained at 37 °C (5 % CO2) in complete RPMI or DMEM respectively (Lonza, Levallois-Perret, France) supplemented with 10 % fetal bovine serum (FBS; Lonza), 5x104 U/L penicillin/streptomycin (Lonza) and 2 mM L-glutamine (Lonza). HeLa and C33-A cells were grown in complete EMEM (Lonza) supplemented with 10 % FBS, 5x104 U/L penicillin/streptomycin and 2 mM L-glutamine.
HPV positive and HPV negative cells were diluted at indicated concentrations in the STM (Digene/Qiagen, Courtaboeuf, France) as reference medium and in the Novaprep® HQ+ Orange medium (Novacyt, Vélizy-Villacoublay, France), an alcohol-based fixative primarily dedicated to liquid-based cytology. Because pretreatment of viscous samples with dithiothreitol (DTT), a mucolytic agent, may be recommended to provide mucus-free single-cell suspensions, Novacyt also provided the Novaprep® HQ+ orange containing 0.03 % DTT. For convenience, the Novaprep® HQ+ medium without DTT will be referred to as HQ + A and the Novaprep® HQ+ medium with DTT will be referred to as HQ + B.
HPV positive and HPV negative cells were diluted at indicated concentrations in the STM (Digene/Qiagen, Courtaboeuf, France) as reference medium and in the Novaprep® HQ+ Orange medium (Novacyt, Vélizy-Villacoublay, France), an alcohol-based fixative primarily dedicated to liquid-based cytology. Because pretreatment of viscous samples with dithiothreitol (DTT), a mucolytic agent, may be recommended to provide mucus-free single-cell suspensions, Novacyt also provided the Novaprep® HQ+ orange containing 0.03 % DTT. For convenience, the Novaprep® HQ+ medium without DTT will be referred to as HQ + A and the Novaprep® HQ+ medium with DTT will be referred to as HQ + B.