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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

Induction of down-regulation of TLR1 expression on the A549 cells.The cells were previously stimulated with different doses of P. aeruginosa lysate (see the text for details) and the TLR1 level was returned to the background by changing the medium to one containing animal serum. The recovery after 24 h was needed to restore the level to equilibria (the left), and the cell numbers in each culture were maintained constant (the inset). The maximum recovery ratio attained during this period was plotted against the stimulation dose of lysate (the right). The TLR signal relative to the background was also graphed. The stimulation dose compromising the two parameters was a 1/100 dilution of the lysate.
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f2: Induction of down-regulation of TLR1 expression on the A549 cells.The cells were previously stimulated with different doses of P. aeruginosa lysate (see the text for details) and the TLR1 level was returned to the background by changing the medium to one containing animal serum. The recovery after 24 h was needed to restore the level to equilibria (the left), and the cell numbers in each culture were maintained constant (the inset). The maximum recovery ratio attained during this period was plotted against the stimulation dose of lysate (the right). The TLR signal relative to the background was also graphed. The stimulation dose compromising the two parameters was a 1/100 dilution of the lysate.

Mentions: Although up-regulation of TLR on the cell surfaces was optimized, monitoring of repetitive stimulations for the same culture requires restoration of the receptor density to levels close to the background (Figure 2, the left). To this end, we investigated stimulation doses that allowed us to rapidly bring the expressed density back to the initial state. The TLR expression level was decreased by eliminating the stimulation source and supplementing the media with components which could protect and detoxify the cells. These conditions were simply achieved by changing the medium to one containing animal serum. The receptor density was then monitored at predetermined times using the immunological assay described above (line graph in the left). During the monitoring of down-regulation, the cell density of each culture was assured to be constant by employing the same staining method described above (bar graph).


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)

Induction of down-regulation of TLR1 expression on the A549 cells.The cells were previously stimulated with different doses of P. aeruginosa lysate (see the text for details) and the TLR1 level was returned to the background by changing the medium to one containing animal serum. The recovery after 24 h was needed to restore the level to equilibria (the left), and the cell numbers in each culture were maintained constant (the inset). The maximum recovery ratio attained during this period was plotted against the stimulation dose of lysate (the right). The TLR signal relative to the background was also graphed. The stimulation dose compromising the two parameters was a 1/100 dilution of the lysate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Induction of down-regulation of TLR1 expression on the A549 cells.The cells were previously stimulated with different doses of P. aeruginosa lysate (see the text for details) and the TLR1 level was returned to the background by changing the medium to one containing animal serum. The recovery after 24 h was needed to restore the level to equilibria (the left), and the cell numbers in each culture were maintained constant (the inset). The maximum recovery ratio attained during this period was plotted against the stimulation dose of lysate (the right). The TLR signal relative to the background was also graphed. The stimulation dose compromising the two parameters was a 1/100 dilution of the lysate.
Mentions: Although up-regulation of TLR on the cell surfaces was optimized, monitoring of repetitive stimulations for the same culture requires restoration of the receptor density to levels close to the background (Figure 2, the left). To this end, we investigated stimulation doses that allowed us to rapidly bring the expressed density back to the initial state. The TLR expression level was decreased by eliminating the stimulation source and supplementing the media with components which could protect and detoxify the cells. These conditions were simply achieved by changing the medium to one containing animal serum. The receptor density was then monitored at predetermined times using the immunological assay described above (line graph in the left). During the monitoring of down-regulation, the cell density of each culture was assured to be constant by employing the same staining method described above (bar graph).

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