A commitment was made to provide evidence for unanswered questions such as the effect of tube composition on assay results.
A theoretical model for describing luminescence kinetics and optimizing composition of the assay detection mixture is presented.
Thus, the plant system is preferable for the CdS biosynthesis because it does not contain a large amount of the macromolecular compounds that affect the composition of the assay sample.
The composition of the assay buffers was as follows: for 5-HT1AR: 50 mM Tris HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 µM pargyline and 0.1% ascorbate; for 5-HT6R: 50 mM Tris HCl, 0.5 mM EDTA and 4 mM MgCl2, for 5-HT7bR: 50 mM Tris HCl, 4 mM MgCl2, 10 µM pargyline and 0.1% ascorbate; for dopamine D2LR: 50 mM Tris HCl, 1 mM EDTA, 4 mM MgCl2, 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate.
The final composition of the assay mixture was 20 mM glucose, 10 mM magnesium acetate, 1 mM ADP, 10 mM AMP, 0.4 mM thio-NAD, 10 mM dithiothreitol, 0.5 U/ml hexokinase, 1 U/ml glucose-6-phosphate dehydrogenase and a range of phosphocreatine concentrations between 0 and 35 mM, all in 0.1 M glycylglycine, pH 6.75.
In these studies, the normal tissue only was irradiated, tumour cells were implanted one day after irradiation, and the composition of AHTs assayed as a function of time post-irradiation.
The final composition of the 20S assay buffer was 250 m m HEPES, 5 m m EDTA, and 0.03% SDS (pH 7.5).
The final composition of the 26S assay buffer was 50 m m Tris, 1 m m EDTA, 150 m m NaCl, 5 m m MgCl2, 50 μ m ATP, and 0.5 m m dithiothreitol (pH 7.5).
The composition of the CFSE assays was as above except that no cells were irradiated and the responder population was stained with CFSE prior to setup.
Additional file 15: Compositions of enzymatic assays employed in the study.
We also tested the effect of assay plate composition in accordance with other published chemotaxis assays: "Jansen" (20 g/L agar, 5 mM potassium-phosphate (pH = 6.6), 1 mM CaCl2, 1 mM MgSO4 [8]), "Ward" (15 g/L agarose, 10 mM HEPES (pH = 7.2), 0.25% Tween 20 [3]) and "Pierce" (17 g/L agar, 2 mM NH4Cl, 1 mM CaCl2, 1 mM MgSO4, 25 mM potassium-phosphate (pH = 6.5) [28]).
