A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria

Abstract

BackgroundQuantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells.ResultsHerein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99.ConclusionsThe ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.

In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

“…No. #CT602) (1:1000 dilution) combined with a secondary goat anti-mouse infrared (IR) Dye 680RD antibody (Licor Biosciences, US) (1:2000 dilution), as previously described [ 22 ]. Briefly, following permeabilization with 0.1% triton X-100 in PBS for 8 min at Room Temperature (RT), cell monolayers were incubated with Odyssey Blocking Buffer for 30 min at RT.…”

“…To overcome such issues, a novel and fast approach, that utilizes near-infrared laser-based scanning, namely the In-Cell Western assay (ICW), has been proposed for viruses and intracellular bacteria, including C . trachomatis [ 22 ]. The ICW assay is a cell-based technique for intracellular protein detection and has been largely exploited for the quantitative analysis of cellular signaling pathways due to its high accuracy and reproducibility [ 23 ].…”