Immunofluorescence Centrosome Staining of Chlamydia-Infected Cells

Immunofluorescence centrosome staining was done as previously established with modification (20 (link), 46 (link)). HeLa, A2EN, or primary cervical cells were infected with the appropriate strains of C.t. at an MOI of 1 by centrifugation at 700 × g for 30 min. At 36 h postinfection, cells were fixed on ice with cold methanol for 6 min and blocked for 2 h at room temperature in 0.1% Triton-X in PBS with 2% FBS. Cells were stained with anti-pericentrin (abcam) and anti-Chlamydia HSP60 (Millipore Sigma). Dylight-488 and Dylight-594 (Thermo Fisher Scientific) secondaries were used along with DAPI (Thermo Fisher Scientific) to stain the nuclei. Images were captured using a Leica DFC7000T confocal microscope equipped with Leica software. At least 10 images were collected per coverslip, with three technical replicates per biological replicate, with at least 2 biological replicates.