Figure 2

Determination of the specificity of the continuous assay for the measurement of TF PCA. (a) Progress curves for the continuous assay performed on T24/83 cells showing response of vehicle control vs 10 μM thapsigargin (Tg), 10 μM thapsigargin pretreated with 10 μg/ml anti-TF neutralizing antibody (Tg + anti-TF) and 10 μM thapsigargin pretreated with 10 μg/ml anti-GFP antibody (Tg + anti-GFP) as a control for the specificity of the TF neutralizing antibody effect. (b) Maximum rate of kinetic reaction (Vmax) derived from progress curves on T24/83 cells showing significant increases in Vmax produced by 10 μM Tg and 10 μM ionomycin (iono) treatment (^*P<0.05 vs vehicle control), significant inhibition of this effect by TF neutralizing antibody (‡P<0.05 vs agonist, Tg and Iono) and no significant effect on Vmax of nonspecific antibody control, anti-GFP. (c) TF PCA of T24/83 cells pre-treated with the anti-TF neutralizing antibody (anti-TF, 1–10 μg/ml) followed by treatment with agonist ionomycin (10 μM), hydrogen peroxide (10 mM) or thapsigargin (10 μM). ^*P<0.05 vs vehicle control. ^** represent significant decrease in agonist response due to pretreatment with anti-TF neutralizing antibody, P<0.05. (d) On cell controls using T24/83 cells where each component of the cocktail is absent without stimulation or with ionomycin (10 μM) or thapsigargin (15 μM) treatment. ^*P<0.05 vs vehicle control (‡ over 0).