We measured the slope of the straight line at steady state, typically between 100 and 600 sec after addition of GTP.
Flow cytomeric analysis began 60 sec before addition of pMVs and continued for 720 sec afterward; FACS analysis was carried out with a LSR II flow cytometer equipped with a temperature-control device.
The merocyanine at a final concentration of 1.35 µM was supplemented to sperm suspension; the subsample was then analyzed directly on the flow cytometer (BD-FACScalibur) about 90 sec after addition of merocyanine.
To one gram of intestinal content, pooled per segment, were added 100 μL of the IS working solution followed by a vortex mixing step (15 sec) and addition of 3 mL PBS and 5 mL ethyl acetate.
Conventional PCR using primers distinguishing MUC1-TM from a secreted isoform of MUC1 which lacks the transmembrane region, MUC1-SEC, demonstrated that approximately half of the samples from malignant mesothelioma cells either from solid tumour or from effusions expressed MUC1-SEC in addition to the transmembrane form.
Fluorescence responses from 20∼30 sec after tastant addition were measured, corrected for background fluorescence measured before compound addition, normalized to the response to 10 µM ISO.
Measurements of increase in absorbance at 234 nm for 30-90 sec after enzyme addition were done in 0.2 M borate buffer (pH 7.5) with linoleic acid (134 μM) as substrate and an enzyme concentration of 167 U/mL, using test substance solutions in DMSO or water or (for blanks) DMSO or water alone.
Mitochondrial light scattering changes 40 sec after the addition in the experimental buffer were used to generate the data shown in the figure.
The decrease in relative change in fluorescence intensity (ΔF/F) at 300 sec after CCCP addition was considered as the relative measure of MMP.
This period of steep decrease of the RFP-PH signals at the plasma membrane corresponds to 80 100 sec after the addition of sperm, and the result is in general agreement with the timing of the accelerated InsP3 production observed in the fertilized eggs of the sea urchin species such as A. crassispina, H. pulcherrimus, and L. pictus [29], [32].
