Journal:

Article Title: Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ib?

doi: 10.1042/BJ20041836

Figure Lengend Snippet: (A) Surface expression of GPIbα was analysed by flow cytometry as described in the Materials and methods section. GPIbα expression was similar for all cell lines; WT (grey-filled histogram), W570A (thick grey line), FW-AA, (black dotted line), F568A (thin black line) and R572A (thick black line). (B) Cells were perfused through BvWf-coated microslides (10 μg/ml) at a shear stress of 0.1 Pa. After 5 min of perfusion, the mean number of adherent cells/field over 5 fields was calculated and the results are expressed as the number of adherent cells/field (means±S.E.M.) for four to seven experiments.

Article Snippet: Flow-based adhesion assays were performed as described previously using glass microslides (VitroCom, NJ, U.S.A.) coated with 10 μg/ml BvWf (bovine vWf) [ 19 ].

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ib?

doi: 10.1042/BJ20041836

Figure Lengend Snippet: CHO cells were perfused through BvWf-coated microslides (10 μg/ml) for 5 min at a shear stress of 0.1 Pa, followed by stepwise increases in shear stress up to 6 Pa. (A, B) Cell-rolling velocities were analysed as described in the Materials and methods section and the results presented are the means±S.E.M. for four to seven experiments. At 0.5 Pa there were no significant differences in rolling velocities between WT and any of the mutant cell lines, whereas at 6 Pa, only W570A and FW-AA cells exhibited significantly faster velocities when compared with WT (P<0.01 and 0.001 respectively). (C, D) Cell detachment was analysed in the same experiments as described in the Materials and methods section and the results are presented as means±S.E.M. There was essentially no detachment of any of the cell lines at 0.5 Pa, but at 6 Pa both W570A and FW-AA were significantly less capable of remaining adherent (P<0.01 in both cases) when compared with WT. There was no significant difference between the adhesion of F568A and R572A cells compared with WT, for both rolling velocity and their ability to remain adherent at high shear stress.

Article Snippet: Flow-based adhesion assays were performed as described previously using glass microslides (VitroCom, NJ, U.S.A.) coated with 10 μg/ml BvWf (bovine vWf) [ 19 ].

Techniques: Mutagenesis

Journal: Cell calcium

Article Title: DETERMINING CALCIUM CONCENTRATION IN HETEROGENEOUS MODEL SYSTEMS USING MULTIPLE INDICATORS

doi: 10.1016/j.ceca.2007.02.002

Figure Lengend Snippet: We measured the average fluorescence intensity in a system consisting of two overlapping 20 μm thick microslides (s=0.5) (A, inset) containing 0 and 1 mM free calcium (A) or, nominally, 0.1 and 10 μM (B) free calcium. Upon collecting the fluorescence of several indicators (fura-2 (F-2), fura-5F (F-5F), fura-4F (F-4F), fluo-3FF (FL3FF), fura-6F (F-6F) and fura-2FF (F2FF) from the ROI (A, inset, dashed box), we calculated [Ca2+]obs using Eq. 8 and 9 [1] as if the fluorescence were collected from a uniform system. As a reference, we marked the nominal free calcium concentrations in both compartments and their average (dotted lines). The data were derived from 2–4 independent experiments.

Article Snippet: We built an in vitro model system consisting of two microslides with 20 μm interior depth (Vitrocom, Mountain Lakes, NJ) intersecting at a 90° angle (see inset in ).

Techniques: Fluorescence, Derivative Assay