TABLE 4.
Comparison of DChol (green) colocalization with different fluorescent membrane markers (red)
| Colocalization Coefficients |
||||
|---|---|---|---|---|
| Fluorescent PM Markers | Mander's |
Costes Randomization (N = 200) % (Robs > Rrand) | Observed/Random Ratio (mean ± SE) | |
| Mred | Mgreen | |||
| DHE (n = 22) | 0.61 | 0.56 | 100% | 2.28 ± 0.08 **, # |
| DiD (n = 23) | 0.51 | 0.50 | 100% | 1.93 ± 0.14 *, # # |
| Alexa Fluor CT-B (n = 26) | 0.59 | 0.30 | 100% | 2.19 ± 0.13 **, # |
| BC-θ (n = 14) | 0.59 | 0.27 | 100% | 2.69 ± 0.21 ** |
| N-Rh-DOPE (n = 16) | 0.29 | 0.20 | 0% | 1.49 ± 0.10 |
DChol and a membrane domain probes were collected in two separate channels. The images were background corrected, threshold was set to select pixels with above average intensities, and regions of interests were drawn to include only the plasma membrane of each cell. Mander's colocalization coefficients Mred and Mgreen were calculated and Costes Randomization Test was performed with ImageJ using Colocalization Test plugin as described in supplementary Method IV. The observed/random ratio was the ratio of the observed percentage of colocalized pixels to the predicted percentage of colocalized pixels assuming a random distribution of both probes. *, **, Significantly different from that of N-Rh-DOPE; #, ##, significantly different from that of BCθ; *, #, P < 0.05; **, # # P < 0.01.