Odyssey In-Cell Western for quantification of Chlamydia trachomatis and screening of anti-chlamydia drugs

Chlamydia trachomatis is the leading infectious cause of blindness among the world's poor and one of the major bacterial sexually transmitted pathogens in developed countries, with approximately 127 million new infections each year

Over the years, several methods have been used to measure chlamydia infection, such as cell culture, direct immunofluorescence assay, RT-PCR

Cell culture has long been considered the "gold standard" for chlamydia drug resistance detection due to its high specificity (100%) and its ability to isolate clinical chlamydia strains

To overcome these difficulties, a novel and rapid method has been proposed for virus and intracellular bacterial detection: In-Cell Western (ICW) detection based on near-infrared laser imaging

In this paper, the authors describe in detail ICW detection by Odyssey CLx as a rapid, more accessible, high-throughput platform for quantification of Chlamydia trachomatis and screening of anti-Chlamydia drugs

Quantitative detection of IFUs of Chlamydia trachomatis by ICW method

To evaluate the performance of the Odyssey CLx instrument using the ICW method for the detection of C.

(A) Near-infrared scanning images of chlamydia-infected cells in standard polystyrene cell culture and optically clear-bottom microplates; (B) signal-to-noise ratio of chlamydia-infected cells in standard polystyrene cell culture and clear-bottom microplates; According to the near-infrared absorbance data with or without background (C) or no background (D), the standard curve of Chlamydia trachomatis IFU was calculated by ICW method on optically transparent bottom and standard microplate, which was similar to the DFA method to count Chlamydia IFU; in ( E) Linear regression model of the standard curve determined by ICW method after logarithmic transformation on standard polystyrene cell culture microplates or (F) clear bottom microplates

In addition, the customer determined the IFU value of Chlamydia trachomatis in unknown samples by ICW and DFA, and the results showed that the ICW measurement value (IFU/mL) of the standard microplate and clear microplate was lower than the DFA value (IFU/mL) (p = 0.

Determination of the sensitivity of Chlamydia trachomatis to erythromycin by ICW

To evaluate the performance of the ICW method as a new method for screening anti-chlamydia drugs, the authors selected erythromycin as the recommended antibiotic for the treatment of Chlamydia trachomatis infection and compared the DFA method

(A) ICW and DFA assays as a function of NIR absorbance of Chlamydia IFU and erythromycin concentration; (B) Infection treated with erythromycin corresponding to MIC (0.

The results of this article show that the ICW method can be used as a simple, economical and rapid alternative to DFA for the quantitative detection of chlamydia inclusion bodies and the sensitivity detection of anti-chlamydia drugs

As a quantitative immunofluorescence (IF) assay method developed by LI-COR in the United States, ICW has been widely used in autophagy, apoptosis, cancer, drug research, signal transduction, ion channels, Gene expression, virological detection, cell proliferation experiments, etc.

Agilent cell culture imaging microplates are
ultra-thin at the bottom, with bottom-reading detection function, high-resolution imaging, and can be widely used in ICW/high content imaging/live cell imaging/microscopic imaging and cell culture-based fluorescence and chemiluminescence experiments
.

Blocking
Buffers LI-COR offers a variety of blocking buffers for use with the Odyssey imaging system
.
Intercept's non-mammalian protein-based blocking buffers (#927-60001, 927-70001) are optimized for use with IRDye® products and other near-infrared fluorophores
.
They provide excellent performance, low variability, and low background for quantitative western blotting and other immunoassays
.

CST IF
Primary Antibody ICW is a quantitative immunofluorescence assay performed in microplates (optimized for 96-well or 384-well format), and an IF-validated primary antibody is recommended
.
CST is committed to providing highly specific antibodies that generate a strong specific signal while minimizing background, and offers more than 1000 antibodies that can be used in ICW assays
.
As a high-quality first-class agent of CST, Gene Co.
, Ltd.
provides better support and assistance for your experiments
.

LI-COR IRDye near-infrared fluorescent secondary antibodies
RDye series of near-infrared fluorescent dyes have been optimized for the Odyssey ® series of imaging systems, featuring high brightness and good photostability
.
In addition, the IRDye series of near-infrared fluorescent dyes have a narrow emission spectrum and are pre-treated with high cross-linking adsorption, which has a very high signal-to-noise ratio, and the cross-reaction has been minimized, making it suitable for multi-color detection
.

CellTag™ Stain
As a solution for ICW Assay homogenization, CellTag stain accumulates in the nucleus and cytoplasm of permeabilized cells and provides a linear fluorescent signal in a variety of cell types and cell numbers
.
This method is fast and simple, and can be combined with secondary antibody incubation
.

Empiria Studio software
is developed in cooperation with top journals such as Nature: powerful validation, analysis and reporting functions perfectly meet all journals' requirements for high-quality data (from raw image acquisition to reproducible analysis)
.

As an agent of LICOR products in China, Gene Co.
, Ltd.
provides you with professional near-infrared imaging technology solutions in Western Blot, In-Cell Western, two-color EMSA, in vivo imaging, etc.
, and provides more application consulting and technical support
.
For more details, please pay attention to our official WeChat "Gene Express" or contact the staff of Gene Co.
, Ltd.
near you
.

Reference
Simone F.
, et.
al.
(2021).
In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials.
PLoS One.
2021; 16(5): e0251075.
PMID: 33974662