Technical Reference #285

285.

Fluorescence Lifetime-Resolved pH Imaging of Living Cells Hai-Jui Lin; Petr Herman; and Joseph R. Lakowicz, University of Maryland, Cytometry, 52(285), (2003)
Link To Paper

Abstract:
Background: The regulation and maintenance of intracellularpH are critical to diverse metabolic functions of theliving cells. Fluorescence time-resolved techniques andinstrumentations have advanced rapidly and enabled theimaging of intracellular p

Keywords:
fluorescence lifetime imaging microscopy; pH imaging; cytosolic pH; lysosomes; DM-NERF; Oregon Green 514 carboxylic acid; carboxy-SNAFL2

Materials & Methods:
Three adherent cell lines were raised for FLIM measurements. 3T3-Swiss albino (CCL-92 ATCC Manassas VA) was grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% bovine calf serum. CHO-K1 (CCL-61 ATCC) was grown in Ham’s F-12K medium with 10% fetal bovine serum. MCF-7 (HTB-22 ATCC) was grown in Eagle’s Minimum Essential Medium containing Earle’s balanced salt saline nonessential amino acids bovine insulin and 10% fetal bovine serum. All media sera and supplements for cell cultures were purchased from Life Technologies (Rockville MD). The cells were routinely cultured at 37°C in 5% CO2 and plated onto glass-bottom microwell dishes (Mattek Corp. Ashland MA) 2 to 3 days before FLIM measurements. Cell Labeling Fluorescent pH indicators were obtained from Molecular Probes (Eugene OR.) without further purification. The stock solution of C-SNAFL2 diacetate in dimethyl sulfoxide (DMSO) was prepared at the concentration of 12 mM and kept desiccated at 20°C. The final working concentration of C-SNAFL2 diacetate was 15 M in Hank’s balanced salt solution (HBSS). Cells were rinsed with HBSS and then incubated with C-SNAFL2 diacetate at 37°C for 20 to 40 min. C-SNAFL2 diacetate diffused across the cell membranes spontaneously hydrolyzed into C-SNAFL2 by intracellular esterase and localized in the cytosol. The extracellular probes then were rinsed off with HBSS. The loaded C-SNFAL2 stayed inside the cells for at least 2 h when the acquisitions were performed at the room temperature. Raising the incubation temperature to 37°C increased cell activities and hastened the release of C-SNAFL2 to the extracellular medium. Therefore the intensified background degraded the resolution of the lifetime images. The lysosomes were stained with the fluorescent probes by spontaneous pinocytosis of the fibroblasts. Dextran- conjugated DM-NERF (dx-DMNERF) and OG-514 carboxylic acid (dx-OG514) of 70 kD were dissolved at the concentration of 1 mg/ml in HBSS and then incubated with the cells at 37°C for 2 h. Afterward the cells were gently rinsed with clean HBSS and incubated at 37°C for 1 h. The second incubation step exhausted the fluorophores kept inside the recycling endosomes and avoided the background increase during the measurements. Lyso- Sensor Yellow/Blue DND-160 (DND-160) is a membranepermeable probe and can rapidly highlight the acidic compartments within 5 min. Nevertheless DND-160 also leaked out the cells in a short time and quickly increased the image background. To obtain better lysosomal images we also used the longer incubation procedures originally used for the dextran-conjugated probes to load DND-160 into cells.

Microscopic Technique
Fluorescence Microscopy, Fluorescence Lifetime Resolved Imaging

Cell Type(s)
CHO