Chlamydia trachomatis Immunofluorescence Assay

Immunofluorescence staining of formaldehyde-fixed cells (2%, 20 min) in 96-well plates was conducted as described previously [57 (link)]. A rabbit-anti-Slc1 serum (1:400, described elsewhere [58 (link)]) in combination with AlexaFluor555- or AlexaFluor647-labeled secondary antibodies (Thermo-Fisher-Scientific) was used for the detection of C. trachomatis. When indicated, DNA was stained for 10 min with 2–10 µg/ml Hoechst 33342 (Thermo-Fisher-Scientific) and cells were stained for 10 min with either 5 µM CellTrace CFSE (Thermo-Fisher-Scientific) or 2 μg/ml HCS CellMask Deep Red stain (Thermo-Fisher-Scientific). Images were acquired on an ImageXpress Micro XL system (Molecular Devices) or on a Cellomics ArrayScan VTI HCS imaging system (Thermo-Fisher-Scientific). Automated image analysis (such as for the determination of the percentage of infected cells, inclusion numbers, or average fluorescence intensities per cell) was conducted using MetaXpress 5.3.0.4 (Molecular Devices) or HCS Studio Cell Analysis Software (Thermo-Fisher-Scientific), respectively.