The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. To quantify the uncertainty of dissociation rate determination, the standard deviation and of arrests with duration within time intervals [0.15 s, 0.45 s] and [0.45 s, ], respectively (Pierres et al., 2002), yielding the formula: (4) The fraction of particles remaining bound at time one second was obtained by dividing the number of arrests lasting 1 s or more (obtained by extrapolation to time zero of the dissociation plot (see Results section). The standard deviation for parameter = was calculated as prescribed by Snedecor and Cochran (1980): (5) FIGURE 2 Duration of cell-fibronectin association. Monocytic THP-1 cells moving along fibronectin-coated surfaces displayed binding events with a wide range of durations. This figure shows a typical detachment curve obtained after recording 448 arrests on 1226 … The aforementioned formulae were used to assess the significance of differences between two experimental dissociation plots obtained under different experimental conditions by applying a standard chi-square test (with two degrees of freedom) to the following sum of squares: (6) Note that the limiting values of bonds at time is Kronecker’s symbol. It was recently found (Pierres et al., 2002) that better ADX-47273 fit between experimental and calculated plots might be obtained with the so-called multiple-bond model with instantaneous bond formation: following this model, represents an average bond number ADX-47273 that will be referred to as Poisson parameter. ADX-47273 The underlying assumption is that several bonds may be formed within less than a fraction of a second if several ligand-receptor couples are at binding distance when cell-surface contact occurs. Second, it is now well documented that a single ligand-receptor complex may undergo different states with different lifetimes (Pierres et al., 1995, 2002; Merkel et al., 1999). The simplest way of accounting for this possibility consisted of assuming that each cell arrest resulted from the formation of a transient complex with dissociation rate transient-state interactions and stabilized interactions at time is: (10) where is the proportion of type-A complexes, which should range between 0 and 1. Note that this minimal model uses three fitted parameters. Assessment of receptor aggregation with confocal microscopy Data acquisition Receptor aggregation was assessed semiquantitatively by taking advantage of the exquisite sensitivity of confocal microscopy. Indeed, this device was found to detect a few or even single fluorescent molecules (Nie et ADX-47273 al., 1994). Under standard conditions, cells were labeled in the cold with fluorescein-conjugated anti-CD29 mouse monoclonal antibodies as previously described, with or without a second layer of unlabeled polyclonal goat anti-mouse immunoglobulin. They were then fixed with 1% paraformaldehyde and examined rapidly with a confocal laser fluorescence microscopy (Leica CLSM, Leica Microsystems, Heidelberg, Germany), using an Argon/Krypton laser (Omnichrome, Leica) and a 40 dry objective. Pixel size was thus 245 245 nm2 with a vertical resolution of order of 700 nm. Typically, a given cell was represented as a series of about six 512 512 pixel images (8-bit depth) with 2-such that 50% of fluorescent molecules are located in pixels with a brightness lower than is the background fluorescence intensity, as measured on a buffer solution, and of a cell incubated in a solution of fluorescent ligand of concentration [as the total number of binding sites on the cell, as the intrinsic fluorescence of a ligand molecule, and ? was approximated as the limit of (? ? were then determined with the Scatchard procedure by plotting 1/{(? suggest that no bond formation occurred after arrest at low-fibronectin density, because detachment curves found on surfaces treated with 1 < 0.05). The fitted parameters were 0.25) than the continuous bond-formation model (< 0.10). It may also be noticed that only the immediate bond-formation model yielded Rabbit polyclonal to ZBTB6. downward concavity of the initial part of detachment curves. FIGURE.