Fig 1.
CoA improved linear range of bioluminescence analysis of ATP in buffer A. Bioluminescence analysis of ATP was performed in buffer A and standard curve was made (A). The effects of CoA on the signal intensity of ATP analysis were evaluated (B). Then standard curve analysis of ATP (C) was made again in the presence of CoA at optimized concentration (15 μM) and signal attenuation of ATP analysis (D) was also estimated by determining relative light unit (RLU) at different time points. Data are representative of three independent experiments. * P < 0.05 vs CoA (15.6 μM).
Fig 2.
Bioluminescence analyses of ATP in HBSS and Tyrode’s buffer have optimized linearity in the presence of CoA.
Bioluminescence analysis of ATP was performed in HBSS and Tyrode’s buffer with or without CoA (15 μM) and standard curves were made (A and B). The signal attenuations of ATP analyses in the presence of CoA (C and D) were also estimated by determining RLU at different time points. Data are representative of three independent experiments.
Fig 3.
BSA slowed down signal attenuations of bioluminescence analyses of ATP in HBSS and Tyrode’s buffer.
Bioluminescence analyses of ATP (1 μM) were performed in HBSS and Tyrode’s buffer in the presence of CoA (15 μM) and serial concentrations of BSA (A and B). The signal attenuations of ATP analyses in the presence of serial concentrations of BSA were also estimated by RLU80min/RLU5min (C and D). Then in the presence or absence of BSA (0.05%), standard curve analyses of ATP in HBSS (E and G) and Tyrode’s buffer (F and H) were performed at different time points. Data are representative of three independent experiments. * P < 0.05 vs BSA (0.0625%).
Fig 4.
BSA didn’t improve signal attenuation of chemiluminescence analysis of HRP and OVA slowed down signal attenuation of bioluminescence analysis of ATP.
In the presence or absence of BSA (0.05%), chemiluminescence analysis of HRP activity (0.001 U/ml) was performed in enhanced chemiluminescence solution at different time points (A) and signal attenuation was evaluated by RLU20min/RLU5min (B). Bioluminescence analyses of ATP (1 μM) were carried out in HBSS and Tyrode’s buffer with or without OVA (0.05%) at different time points (C and E). The signal attenuations of ATP analyses were analyzed by RLU80min/RLU5min (D and F). Data are representative of three independent experiments. * P < 0.05 vs BSA (0.05%) or OVA (0.05%).
Fig 5.
Method validation of ATP analysis in the presence of platelets and bioluminescence analysis of ATP released by activated platelets.
Washed platelets were suspended in HBSS or Tyrode’s buffer and seeded in white 96-well plate. In the presence of platelets, standard curves of ATP were performed by adding of CoA (15 μM) into reaction system (A and B) and signal attenuation curves were analyzed by adding of BSA (0.05%) into reaction system (C and D). Data are representative of three independent experiments. Recovery rates of ATP analyses in HBSS and Tyrode’s buffer were analyzed by adding standard ATP (0.1μM or 1μM) into reaction system (E and F). Data are from three independent experiments. At last, the platelets were added into 96-well plate or 384-well plate, and then stimulated with thrombin or collagen at 37°C for 20 min. Bioluminescence analysis of ATP released by activated platelets was initiated by adding luciferase-luciferin work solution containing CoA and BSA at optimized concentrations into 96-well plate and RLU values were determined at 5 min after the start of the reaction (G and H). Concentrations of released ATP and Z’ factors were calculated. Data are from five independent experiments. * P < 0.05 vs Control.
Fig 6.
Screening of platelet inhibitors with optimized bioluminescence analysis of released ATP.
Washed platelets resuspended in HBSS or Tyrode’s buffer were seeded in white 96-well plate. Subsequently, the platelets were pretreated with Ly294002 or Staurosporine for 5 min and then stimulated with thrombin at 37°C for 20 min. Bioluminescence analyses of ATP released by activated platelets were performed. Inhibition rates and IC50 of Ly294002 (A and B) or Staurosporine (C and D) on ATP release of activated platelets were calculated. Data are representative of three independent experiments.