Standard curves of C . trachomatis IFUs via ICW assay in optically clear bottom and standard microplates, related to the enumeration of chlamydial IFUs by DFA assay.

ICW assay: confluent McCoy cell monolayers, grown on either 96-well standard polystyrene or optically clear bottom cell culture microplates, were infected with two-fold serial dilutions of C . trachomatis EB suspension, from MOI of 1.0 to 1/2 9 IFUs/cell. After 36 hours post infection, infected cell monolayers were fixed in 4% PFA, permeabilized by 0.1% triton x-100 in PBS, stained and scanned via Odyssey CLx as described in Materials and Methods. DFA: confluent McCoy cell monolayers, grown on coverslips in 24-well cell culture microplates, were infected as above described. After 36 hours post infection, infected cell monolayers were fixed in 96% ice cold methanol, stained and visualized via fluorescence microscopy (400X magnification) as described in Materials and Methods. (A) Representative infrared scan images of Chlamydia-infected cells on standard polystyrene cell-culture or optically clear bottom microplates from at least three independent experiments; (B) Signal to noise ratio of Chlamydia-infected cells in either standard polystyrene cell-culture or optically clear bottom microplates; Standard curves of C . trachomatis IFUs via ICW in optically clear bottom and standard microplates, calculated from near-infrared absorbance data with background (C) or no-background (D) subtraction, related to the enumeration of chlamydial IFU via DFA; Linear regression models of standard curves by the ICW assay after log-transformation on (E) standard polystyrene cell-culture microplates or (F) optically clear bottom microplates. **, p < 0.001; *, p < 0.05.