Technical Reference #1918

1918.

Real-Time Monitoring of Transferrin-Induced Endocytic Vesicle Formation by Mid-Infrared Surface Plasmon Resonance Victor Yashunsky; Simcha Shimron; Vladislav Lirtsman; Aryeh M. Weiss; Naomi Melamed-Book; Michael Golosovsky; Dan Davidov; and Benjamin Aroeti, Hebrew University of Jerusalem, Biophysical Journal, 97(1918), (2009)
Link To Paper

Materials & Methods:
Melanoma cells were cultured to ~50% confluence on Glass Bottom Culture dishes (35 mm dish 14 mm Microwell; MatTek Ashland MA). Cells were first exposed to growth medium lacking serum for 3 h before the experiment and then washed three times with internalization buffer (150 mM NaCl 20 mM Hepes pH 7.4 1 mM CaCl2 5 mM KCl 1 mM MgCl2 10 mM glucose). After the last wash internalization buffer containing 0.1 mMsulfomM sulforhodamine green (SRG) (Biotium Hayward CA) was added to the medium. Cells were imaged with an Olympus FV-1000 confocal microscope equipped with an on-scope incubator (Life Image Services Basel Switzerland) which controls temperature and humidity and provides an atmosphere of 5% CO2. A 60 /NA ¼ 1.35 oil immersion objective was used. Because the anionic SRG does not enter intact cells the cells appear as dark objects against a uniform fluorescent background when imaged with the confocal microscope. First one plane of focus was acquired and then Rhodamine Red-holo Tfn (5 mg/mL; Jackson ImmunoResearch West Grove PA) was introduced into the imaging buffer. Confocal images of both the SRG (Ex: 514 nm; Em: 535–565 nm) and Rhodamine Red holo-Tfn (Ex: 543 nm; Em: 560–660 nm) were acquired from the same section every 10 or 20 s. A similar protocol was used for lissamine rhodamine apo-Tfn except that the SRG was imaged using 488-nm excitation and a 505–525-nm emission filter. The FV1000 was equipped with the Zero Drift Controller option to maintain the same focus plane throughout the entire period of imaging. The images were processed using ImageJ (National Institutes of Health BethesdaMD)as follows. First the despeckle filter (essentially a median filter with a 3 3 kernel) was applied to remove point noise. The SRG image was used to determine the cell boundaries. Then the average fluorescence intensity inside the cells (F(t)intracellular) was divided by the average fluorescence intensity in a region of interest outside the cells (hF(t)iextracellular) i.e. fluorescence signal is Ifluo ¼ F(t)intracellular/F(t)extracellular This procedure was adopted based on the assumption that there is insignificant depletion of labeled Tfn in the medium so that the fluorescence in the medium should remain constant. The original time lapse sequences are provided in Movie S1Movie S2Movie S3 Movie S4 Movie S5 and Movie S6. The ImageJ macros that were used to process the data are available on request from the authors.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Melanoma cells