Purification of Chlamydia trachomatis EBs and RBs

C. trachomatis biovar LGV, serotype L2, strain 434/Bu was propagated in HeLa CCL2 monolayers (ATCC, Rockville, Maryland, USA) grown in Dulbecco's minimal essential medium (DMEM high glucose 1×) (Gibco/Invitrogen Life Technologies, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Mediatech, Inc., Manassas, Virginia, USA) at 37°C, 5% CO₂ in a humidified atmosphere. Infections were carried out in T175 flasks (Sarstedt, Nümbrecht, Germany) by adding a suspension of EBs at a multiplicity of infection (MOI) of 10. C. trachomatis RB and EB forms were collected at 18 and 44 hpi, respectively. Purification of C. trachomatis EBs and RBs was performed by density gradient centrifugation essentially as previously described (Caldwell et al., 1981 (link)) with minor modifications. Briefly, Renografin was replaced by Omnipaque 350 (GE Healthcare, Princeton, New Jersey, USA) supplemented with NaCl 160 mM. The gradients were prepared by diluting Omnipaque 350-160 mM NaCl in SPG buffer (3.8 mM KH₂PO₄, 7.2 mM K₂HPO₄, 4.9 mM L-glutamic acid, 218 mM sucrose, pH=7.4) such that final concentrations of Omnipaque 350 were 28.5%, 38.0%, 41.8% and 51.3% in successive layers. To assure purity of LGV-L2, the strain was plaque purified twice (O'Connell & Nicks, 2006 (link)) before generating EB and RB forms used for proteomic analysis. All strains and cell lines were Mycoplasma -free as determined by a previously described PCR method (van Kuppeveld et al., 1992 (link)).