Inverted widefield microscope

Cells were planted on tissue-culture plastic, allowed to grow to a density of 90%, and serum-starved overnight. Wounds were applied with a pipette tip and washed thoroughly with PBS. Wound closure was thereafter imaged in serum-free medium for the indicated times using an inverted widefield microscope (Nikon). Wound closure efficiency was calculated as percentage of wound area after the imaging (indicated times) compared with imaging starting time. Cell motility was analyzed in more detail by determining the displacement of cells over time and calculating migration speed and directionality as described for the migration assay.

FLIM experiments were performed with a lifetime fluorescence imaging attachment (Lambert Instruments, Leutingewolde, the Netherlands) on an inverted wide‐field microscope (Nikon). Cells plated on poly‐lysine coated Matek dishes and transfected as indicated were excited by using a sinusoidally modulated 3‐W 448‐nm light‐emitting diode at 40 MHz under epi‐illumination. Fluorescein at the concentration of 1 μM was used as a lifetime reference standard (4 ns). Cells were imaged with a 40x Plan‐Apo oil‐immersion objective (numerical aperture 1.45) using a CFP filter set; exposure times were less than 150 ms to avoid photobleaching. The phase and modulation lifetimes were determined from a set of 12‐phase settings using the manufacturer's software (LIFA). FRET efficiency was calculated according to the formula F_eff = 1 − τ2/τ1. FLIM data were averaged on a per‐cell basis. Three experiments (> 40 cells in total) were performed for each condition. Donor alone and donor‐acceptor measurements were taken immediately after one another. In order to prevent differences in donor lifetimes due to altered lipid environments, Cerulean‐TREK‐1 lifetime was measured in the presence of POPDC1 (tagged with Myc or Venus) to enhance PM localization.