Limits...
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.

Show MeSH

Related in: MedlinePlus

Repetitive analysis for cyclic responses of the mammalian cells to repeated stimulations with bacterial lysate.According to the prepared scheme (A), the cellular response to the stimulation increased the TLR level ((B), the left axis) under starvation conditions and then brought the status closely back to the background under appropriate cultivation settings (refer to the stages labeled on the bottom of each graph). This cyclic response could be repeated twice for the same cell culture, where the cell immobilization was carried out for 24 h and stimulation for 4 h each cycle. The TLR level at each stage was converted to the ratio of the density of stimulated cells over that of non-stimulated cells, i.e., signal-to-noise ratio (the right axis), which clearly showed the increasing and decreasing response pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4127502&req=5

f5: Repetitive analysis for cyclic responses of the mammalian cells to repeated stimulations with bacterial lysate.According to the prepared scheme (A), the cellular response to the stimulation increased the TLR level ((B), the left axis) under starvation conditions and then brought the status closely back to the background under appropriate cultivation settings (refer to the stages labeled on the bottom of each graph). This cyclic response could be repeated twice for the same cell culture, where the cell immobilization was carried out for 24 h and stimulation for 4 h each cycle. The TLR level at each stage was converted to the ratio of the density of stimulated cells over that of non-stimulated cells, i.e., signal-to-noise ratio (the right axis), which clearly showed the increasing and decreasing response pattern.

Mentions: The chemiluminescent detection method was applied to monitoring of the regulation switching of TLR1 density in response to repetitive bacterial stimulations (Figure 5). The initial time protocol for each cyclic response was as follows, except pre-cultivation for cell attachment for 24 h (5A): a) basal or down-regulated level detection for 2 h, b) starvation for 2 h, c) stimulation for 4 h under starvation conditions, d) up-regulated level detection for 2 h, and restoration for 18 h using the serum-containing medium. This stimulation-restoration cycle using the same cell culture was repeated two times to repeatedly observe the cellular response regarding TLR expression (5B). Using this protocol, two separate, cyclic responses in the TLR level (the left axis) were observed: an increase upon stimulation under starvation conditions and then a decrease during the restoration stage. For the control, the TLR density of the non-stimulated cell culture was continuously elevated, even if at a negligible rate. The relative value of the two densities at each stage, which is the signal-to-noise ratio (the right axis), explicitly showed an up-and-down response pattern twice, although both the maximum and minimum values in each cycle were slightly increased. The increased rate was 16% for the maximum at up-regulated state by stimulation, and 23% for the minimum at down-regulated state by restoration.


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)

Repetitive analysis for cyclic responses of the mammalian cells to repeated stimulations with bacterial lysate.According to the prepared scheme (A), the cellular response to the stimulation increased the TLR level ((B), the left axis) under starvation conditions and then brought the status closely back to the background under appropriate cultivation settings (refer to the stages labeled on the bottom of each graph). This cyclic response could be repeated twice for the same cell culture, where the cell immobilization was carried out for 24 h and stimulation for 4 h each cycle. The TLR level at each stage was converted to the ratio of the density of stimulated cells over that of non-stimulated cells, i.e., signal-to-noise ratio (the right axis), which clearly showed the increasing and decreasing response pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Repetitive analysis for cyclic responses of the mammalian cells to repeated stimulations with bacterial lysate.According to the prepared scheme (A), the cellular response to the stimulation increased the TLR level ((B), the left axis) under starvation conditions and then brought the status closely back to the background under appropriate cultivation settings (refer to the stages labeled on the bottom of each graph). This cyclic response could be repeated twice for the same cell culture, where the cell immobilization was carried out for 24 h and stimulation for 4 h each cycle. The TLR level at each stage was converted to the ratio of the density of stimulated cells over that of non-stimulated cells, i.e., signal-to-noise ratio (the right axis), which clearly showed the increasing and decreasing response pattern.
Mentions: The chemiluminescent detection method was applied to monitoring of the regulation switching of TLR1 density in response to repetitive bacterial stimulations (Figure 5). The initial time protocol for each cyclic response was as follows, except pre-cultivation for cell attachment for 24 h (5A): a) basal or down-regulated level detection for 2 h, b) starvation for 2 h, c) stimulation for 4 h under starvation conditions, d) up-regulated level detection for 2 h, and restoration for 18 h using the serum-containing medium. This stimulation-restoration cycle using the same cell culture was repeated two times to repeatedly observe the cellular response regarding TLR expression (5B). Using this protocol, two separate, cyclic responses in the TLR level (the left axis) were observed: an increase upon stimulation under starvation conditions and then a decrease during the restoration stage. For the control, the TLR density of the non-stimulated cell culture was continuously elevated, even if at a negligible rate. The relative value of the two densities at each stage, which is the signal-to-noise ratio (the right axis), explicitly showed an up-and-down response pattern twice, although both the maximum and minimum values in each cycle were slightly increased. The increased rate was 16% for the maximum at up-regulated state by stimulation, and 23% for the minimum at down-regulated state by restoration.

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