Crl 1696

C.t. SvA/HAR-13, SvC/TW-3, SvH/UW-43/Cx, SvI/UW-12/Ur, SvJ/UW-36/Cx, SvK/UW-31 SvD/UW-3/Cx, SvE/Bour, SvF/IC-Cal-3, SvG/UW-57/Cx (all from ATCC), SvIa/sotonIa3/Ia870 (from the Chlamydia Biobank), and SvA/2497 and SvB/Tunis-864 (from LSHTM) were propagated in Hela 229 cells (ATCC^® CCL-2™) or McCoy cells (ATCC® CRL-1696™) and harvested by repeated centrifugation and sonication steps. Finally the bacterial suspension was layered on a 30% renografin solution and centrifuged at 40,000 × g for 30 min. After centrifugation, the pellet was resuspended in a sucrose–phosphate–glutamate (SPG) buffer and stored at −80 °C. Serovar typing of the bacteria was confirmed by chromosomal DNA extraction, PCR amplification, and sequencing of the gene and flanking regions of ompA. All C.t. serovars were tested negative for mycoplasma (Mycoplasma laboratory SSI). IFU of the serovar batches was quantified by titration in McCoy cells. Protein concentrations were determined by bicinchoninic acid protein assay (BCA) (Pierce, Thermo Fisher Scientific, Waltham, Massachusetts, US).

McCoy cell line (CRL-1696, ATCC), used as the host cells for the cultivation of C. trachomatis D/UW-3/CX (VR-885, ATCC), are grown in MEM containing 10% fetal bovine serum in a humidified incubator at 37 °C and 5% CO₂ until 90–100% confluent. The C. trachomatis infected McCoy cells are maintained in a special medium consisting of 90 mL DMEM (Life Technologies), 10 mL fetal bovine serum and 1 μg/mL cycloheximide. Further details on C. trachomatis in vitro cultivation and harvest are described in supplementary information. Aliquots of harvested C. trachomatis were stored in Hank Balanced Salt Solution (HBSS) at −80 °C.