Universal Approach to FRAP Analysis of Arbitrary Bleaching Patterns.

Cell Culture

All reagents were purchased from Biological Industries (Kibbutz Beit Haemek, Israel) unless otherwise noted. Normal mouse fibroblasts (ATCC CCL-1.3) were cultured in 250 ml TC flasks (Cellstar, GrenierBio-One, Frickenhausen, Germany) at 37 °C and 5% CO 2 in Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine, 10% fetal bovine serum and 1% Pen-strep solution. For mounting, flasks were washed with 10 ml of phosphate buffer saline (PBS) and then incubated in 2 ml trypsin EDTA 0.25% at room temperature until detachment from the flask (about 2 min). 7.5 × 10 5 cells were plated on homemade glass bottom (#1 cover-slide) 50 mm diameter Petri dish, in 5 ml medium, and incubated overnight. Staining 0.934 mg of 1,1_-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), specifically DiIC 18 (3) (Molecular probes, Eugene, Oregon, USA) were dissolved in 1 mL of Ethanol to prepare a 1 mM Stock solution. For staining of cells, a 2.5 μM staining solution was prepared by adding 2.5 μL bulk solution to 1 mL serum-free growth medium. Following overnight incubation, growth medium was removed from Petri dishes and 500 μL of DiI staining solution were added, followed by 1 hour incubation at 0 °C. The dishes were then washed 3 times with 5 ml normal growth medium, allowing for 5 minutes incubation at 37 °C and 5% CO 2 between washes. Before imaging, growth medium was removed and dishes were gently rinsed 3 times with 5 ml warm (37 °C) Hepes Hank’s balanced salt solution.

Imaging

Cells were imaged with a Hamamatsu C9100-50 cooled EM-CCD camera on an Axiovert-200 M microscope (Zeiss, Germany) with Plan-Neofluar 63x/NA 1.4 objective. The microscope is fitted with a UltraView ERS FRET-H- Spinning Disc Confocal system (Perkin-Elmer, Waltham, MA, USA) with a FRAP module. DiI was bleached using the 488 nm laser line from an Argon ion laser, with varying bleaching time (depending on bleaching pattern and cell intensity).