TABLE 2.
Single-point time-resolved fluorescence polarization anisotropy of diI-C18 and A488-IgE in RBL cells, with and without IgE-FcɛRI cross-linking by α-IgE
| φ1/ns | β1 | φ2/ns | β2 | r∞ | r0 | r∞/r0 | n | |
|---|---|---|---|---|---|---|---|---|
| diI-C18 labeled cells | ||||||||
| − α-IgE | 0.10(1) | 0.12(3) | – | – | 0.21(4) | 0.33(2) | 0.64(9) | 7 |
| + α-IgE | 0.13(1)* | 0.10(3) | – | – | 0.19(7) | 0.30(6) | 0.64(2) | 7 |
| A488-IgE labeled cells | ||||||||
| − α-IgE | 0.13(2) | 0.10(3) | 1.56(2) | 0.07(2) | 0.08(3) | 0.18(5) | 0.45(9) | 13 |
| + α-IgE | 0.16(3)* | 0.07(1) | 1.83(5)* | 0.07(1) | 0.11(4) | 0.19(4) | 0.59(1)* | 11 |
| Free markers in solution | ||||||||
| Alexa 488 (water‡) | 0.11(6) | 0.33(7) | – | – | – | 0.33(7) | – | 2 |
| A488-IgE (PBS‡) | 0.17(2) | 0.12(3) | 2.01(2)* | 0.06(1) | 0.03(1)† | 0.15(1) | 0.22(3)* | 4 |
| diI-C18 (DMSO) | 0.73(6) | 0.30(1) | – | – | – | 0.30(1) | – | 4 |
| Fluorescein (water‡) | 0.12(1) | 0.29(9) | – | – | – | 0.29(9) | – | 3 |
λex = 480 nm, T ≈ 20°C. The fitting parameters represent an average over a number of cells (n) with the standard deviation in the last digit shown in parentheses to reflect the cell-to-cell variation of the individual fit parameters.
p < 0.05, as determined from unpaired, one-tailed Student's t-tests, indicating a statistically significant difference in the means as compared to cells in the absence of receptor cross-linking.
The time constant associated with the third exponential term is ∼40–100 ns with a preexponential factor that is shown in this case as residual anisotropy (r∞). The uncertainty in this time constant is due to the rather small amplitude and the relatively long rotational time as compared with the excited state lifetime of A488-IgE.
The pH of water is ∼5–5.5, and the pH of PBS is 7.4.