Cellomics arrayscan vti hcs imaging system

The AlexaFluor488 Annexin V/Dead Cell Apoptosis Kit (Thermo-Fisher-Scientific) was adapted for use with adherent cells. Briefly, to each well in a 96-well plate containing treated or control cells in 50 µl growth medium, 50 µl of a 2x staining solution (PI (2 µg/ml) and Annexin V-AlexaFluor488 (1:10) in 2x binding buffer) were added. After 15 min incubation at room temperature, 100 µl 1x binding buffer containing 2 µg/ml Hoechst 33342 were added per well and cells were imaged on a Cellomics ArrayScan VTI HCS imaging system (Thermo-Fisher-Scientific). Fluorescence intensity thresholds for distinction between PI- and Annexin V-positive or negative cells were set so that about 95% of all cells in untreated uninfected control wells were classified as double negative (viable) cells.

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.