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Chemiluminometric immuno-analysis of innate immune response against repetitive bacterial stimulations for the same mammalian cells.

Jeon JW, Cho IH, Ha UH, Seo SK, Paek SH - Sci Rep (2014)

Bottom Line: In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder.The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum.Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio-Microsystem Technology, Korea University, 1, 5-ka, Anam-dong, Seongbuk-Gu, Seoul 136-701, Korea.

ABSTRACT
For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.

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Related in: MedlinePlus

Mammalian cell-based chemiluminescent detection system for bacterial stimulation (A) and the analytical procedure (B).The system was developed to quantitatively measure the TLR expression on the A549 cell surfaces with minimal cell damage. The measurement protocol was shown to be effective to clearly distinguish the stimulation signal from the background.
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f4: Mammalian cell-based chemiluminescent detection system for bacterial stimulation (A) and the analytical procedure (B).The system was developed to quantitatively measure the TLR expression on the A549 cell surfaces with minimal cell damage. The measurement protocol was shown to be effective to clearly distinguish the stimulation signal from the background.

Mentions: To measure the chemiluminescent intensity as a signal, a cooled-CCD camera was installed within a dark space, of which the floor was used as a stage for placing cell culture well plates (Figure 4A). The culture plate was selected as it was black in color and the well bottom was made of thin glass, which resolved a main problem in the luminescence detection, i.e., interference between the wells by light dispersion. When a signal image as an indication of bacterial stimulation was obtained, the bottom was first brought into focus by adjusting the lens under illumination (4B, a). After turning the lighting off, the chemiluminescent signal was measured as an image using the Image J program, and the signal of the non-stimulated negative control was also monitored (4B, b). The color intensity of the image was then increased using the software, in order to add contrast, which clearly showed the light signal converged on the edge of the culture well (4C, c). Each image was digitized by using the same program, and the optical densities were then transferred into a computer program (e.g., MS Excel) and vertically added to construct a graph (4C, d). The signal for the TLR expression on the cell culture was finally measured by integrating the optical densities after subtraction of those of the background. Since such a detected signal was proportional to the stimulation dose, the chemiluminescent immuno-anaysis method was applied to the cyclic TLR monitoring for the same cells in response to repetitive stimulations in the next section. It is notable that the signal detection system can be typically 10 times inexpensive comparing to the conventional device measuring fluorescent dye as label. This comparison was made based on the need of extra instrumentation for the fluorescent detection, comparing to that for the chemiluminescent measurement, such as light source for excitation of the dye and a filter of the emitted light36.


Chemiluminometric immuno-analysis of innate immune response against repetitive bacterial stimulations for the same mammalian cells.

Jeon JW, Cho IH, Ha UH, Seo SK, Paek SH - Sci Rep (2014)

Mammalian cell-based chemiluminescent detection system for bacterial stimulation (A) and the analytical procedure (B).The system was developed to quantitatively measure the TLR expression on the A549 cell surfaces with minimal cell damage. The measurement protocol was shown to be effective to clearly distinguish the stimulation signal from the background.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127502&req=5

f4: Mammalian cell-based chemiluminescent detection system for bacterial stimulation (A) and the analytical procedure (B).The system was developed to quantitatively measure the TLR expression on the A549 cell surfaces with minimal cell damage. The measurement protocol was shown to be effective to clearly distinguish the stimulation signal from the background.
Mentions: To measure the chemiluminescent intensity as a signal, a cooled-CCD camera was installed within a dark space, of which the floor was used as a stage for placing cell culture well plates (Figure 4A). The culture plate was selected as it was black in color and the well bottom was made of thin glass, which resolved a main problem in the luminescence detection, i.e., interference between the wells by light dispersion. When a signal image as an indication of bacterial stimulation was obtained, the bottom was first brought into focus by adjusting the lens under illumination (4B, a). After turning the lighting off, the chemiluminescent signal was measured as an image using the Image J program, and the signal of the non-stimulated negative control was also monitored (4B, b). The color intensity of the image was then increased using the software, in order to add contrast, which clearly showed the light signal converged on the edge of the culture well (4C, c). Each image was digitized by using the same program, and the optical densities were then transferred into a computer program (e.g., MS Excel) and vertically added to construct a graph (4C, d). The signal for the TLR expression on the cell culture was finally measured by integrating the optical densities after subtraction of those of the background. Since such a detected signal was proportional to the stimulation dose, the chemiluminescent immuno-anaysis method was applied to the cyclic TLR monitoring for the same cells in response to repetitive stimulations in the next section. It is notable that the signal detection system can be typically 10 times inexpensive comparing to the conventional device measuring fluorescent dye as label. This comparison was made based on the need of extra instrumentation for the fluorescent detection, comparing to that for the chemiluminescent measurement, such as light source for excitation of the dye and a filter of the emitted light36.

Bottom Line: In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder.The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum.Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio-Microsystem Technology, Korea University, 1, 5-ka, Anam-dong, Seongbuk-Gu, Seoul 136-701, Korea.

ABSTRACT
For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.

Show MeSH
Related in: MedlinePlus