TABLE 1.
Single-point, time-resolved fluorescence lifetime of diI-C18 and A488-IgE in RBL cells with and without IgE-FcɛRI cross-linking using α-IgE
| τ1/ns | α1 | τ2/ns | α2 | τ3/ns | α3 | τfl/ns | n | |
|---|---|---|---|---|---|---|---|---|
| diI-C18 labeled cells | ||||||||
| − α-IgE | 0.08(1) | 0.53(5) | 0.62(5) | 0.35(4) | 2.9(2) | 0.12(2) | 0.60(4) | 10 |
| + α-IgE | 0.13(3)* | 0.45(3)* | 0.75(8)* | 0.40(5)* | 3.1(4) | 0.15(4)* | 0.8(1)* | 11 |
| A488-IgE labeled cells | ||||||||
| − α-IgE | – | – | 1.0(1) | 0.81(2) | 3.4(3) | 0.19(2) | 1.5(2) | 5 |
| + α-IgE | – | – | 1.10(6)* | 0.77(1)* | 3.6(1) | 0.23(1)* | 1.67(4)* | 7 |
| Free markers in solution | ||||||||
| Alexa 488 (water†) | – | – | – | – | 4.1(2) | 1.0 | 4.1(2) | 2 |
| A488-IgE (PBS†) | – | – | 1.0(1)* | 0.52(2) | 3.38(2)* | 0.48(2) | 2.13(3)* | 2 |
| diI-C18 (DMSO) | – | – | – | – | 0.61(3) | 1.0 | 0.61(3) | 2 |
| Fluorescein (water†) | – | – | – | – | 3.77(2) | 1.0 | 3.77(2) | 2 |
| Fluorescein (PBS†) | – | – | – | – | 3.94(1) | 1.0 | 3.94(1) | 2 |
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. All data shown, except for A488-IgE labeled cells, are from single-point decays acquired at the magic angle, using deconvolution with the system response function (FWHM ∼60 ps) as in 2P-FLIM experiments (see text). A comparison was made with the fitting parameters of fluorescence decays calculated from parallel and perpendicularly polarized fluorescence decays (see text).
p < 0.05, as determined from unpaired, two-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 pH of water is ∼5–5.5, and the pH of PBS is 7.4.