Hcx plan apo 63 na 1

For calcein and 5-CF transfer assays, calcein and 5-CF staining and FRAP analysis were performed as previously reported. Calcein and 5-CF were loaded into cells as esterified precursors (calcein acetoxymethylester and 5-CF diacetate acetoxymethylester) (23 (link), 27 (link)). Cell suspensions were spotted onto agar and placed in a custom-built temperature-controlled sample holder with a glass coverslip on top. All measurements were carried out at 30°C. For both calcein and 5-CF, cells were imaged with a Leica HCX Plan Apo 63× NA 1.4 oil immersion objective attached to a Leica TCS SP5 confocal laser scanning microscope as previously described for calcein (23 (link)) with a 488-nm line argon laser as the excitation source. Fluorescence emission was monitored by collection across windows of 500 to 520 or 500 to 527 nm in different experiments with a 150-µm pinhole. After an initial image was recorded, bleaching was carried out by an automated FRAP routine that switched the microscope to X-scanning mode, increased the laser intensity by a factor of 10, and scanned a line across one cell for 0.137 s before reducing the laser intensity, switching back to XY imaging mode, and recording a sequence of images typically at 1-s intervals.