Dmi600b fluorescent microscope

C. trachomatis L2-wild type and L2-ΔCT228 were used to infect HeLa cell monolayers on glass coverslips in 24 well plates (CellTreat Scientific, Pepperell MA) at a MOI of ~0.5 (performed in technical triplicates). At 18 h post-infection supernatants were removed and cells were fixed in cold methanol. Recruitment of host proteins was tested with primary antibody staining to MYPT1 (United States Biological Life Sciences, Salem, MA), phospho (pSer-19)-MLC2 (Abcam, Cambridge, MA), phospho (pTyr419) Src (Millipore Sigma, Burlington, MA), Myosin IIa (ThermoFisher Scientific), Myosin IIb (ThermoFisher Scientific), and phospho (pTyr 471) Myosin Light Chain Kinase (Santa Cruz). Chlamydiae were detected with anti-Chlamydia LPS (ThermoFisher Scientific). Fluorescent secondary antibodies, anti-mouse or anti-rabbit DyLight 594 and DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for indirect immunofluorescence. The entire experiment was repeated on three separate occasions (n = 3 biological replicates). Twelve images were captured per condition using a Leica DMI600B fluorescent microscope and a representative image was selected for presentation.

Chlamydia trachomatis L2-wild type and L2-ΔCT228 were used to infect HeLa cell monolayers at 90% confluency in 24 well plates at a MOI of ~0.5; inoculated plates were centrifuged for 1 h at 700 × g then any free EBs were washed away prior to feeding cells. At 0, 6, 12, 24, and 48 h post-infection cells were lysed with water, lysates were serially diluted in Hank's Balanced Salt Solution and used to infect HeLa cells in 96 well plates. At 24 h post-infection, monolayers were fixed in methanol and stained with rabbit anti-EB antibody followed by anti-rabbit DyLight 488 (Jackson ImmunoResearch Laboratories, Westgrove PA). The number of inclusions per field of view were counted in 20 fields for each sample using a Leica DMI600B fluorescent microscope. Total Infectious Forming Units (IFUs)/ml were calculated for each sample. Titers for both the C. trachomatis L2-wild type and L2-ΔCT228 were repeated 3 times to ensure 5 μL contained 1 × 10⁶ EBs.