3rd order polynomial fit  (MathWorks Inc)

Journal: Cell calcium

Article Title: Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor

doi: 10.1016/j.ceca.2009.06.003

Figure Lengend Snippet: FRET analysis of intracellular calcium level with TN-XXL in HEK293T cells. Cells were perfused with 40 mM glucose (A-F). Open bars with gray columns indicate the bolus of external glucose during perfusion with glucose-free Hanks’ balanced buffer. Quantitative data were derived by pixel-by-pixel integration of the ratiometric images. The fluorescence intensity in arbitrary units (A.U.) for individual eCFP (ET470/24m) and Citrine (ET535/30m) emission channels were monitored with eCFP excitation (ET430/24x) and the FRET index for Venus and eCYP was determined (Y-axis corresponds to sensitized fluorescence Fc (background and bleed-through corrected using Citrine excitation (ET500/20x)) normalized to donor emission; note that peak intensities were used and that only the short wavelength emission peak of eCFP was considered for donor emission). FRET images were acquired every 5 sec and cytosolic calcium levels were analyzed. HEK293T cells were preincubated with 50 μM gadolinium (30 min) (B), 10 μM nifedipine (30 min) (C), 1 μM thapsigargin (15 min; D, 30 min; E) or no calcium (30 min) (F) without glucose. When steady-state calcium values were reached, a bolus of 40 mM glucose was added for 2 min. Data are shown as mean ± SD (n = 5-12).

Article Snippet: The baseline of the recordings was corrected using a 3 rd order polynomial fit of the FRET index measured in the absence of glucose in Matlab (the script was programmed by Dr. Oliver Schweissgut, http://www.uni-siegen.de/fb11/simtec/software/fret/ ).

Techniques: Derivative Assay, Fluorescence