Technical Reference #1606

1606.

In vitro analysis of cell metabolism using a long-decay pH-sensitive lanthanide probe and extracellular acidification assay James Hynes a; Tomás C. O’Riordan a; Alexander V. Zhdanov b; Georg Uray c; Yvonne Will d; Dmitri B. Papkovsky, University College Cork, Analytical Biochemistry, 390(1606), (2009)

Abstract:
Metabolic perturbations play a critical role in a variety of disease states and toxicities. Therefore knowledgeof the interplay between the two main cellular ATP generating pathways glycolysis and oxidativephosphorylation is particularly informativ

Keywords:
fluorescent pH-sensitive lanthanide chelate; time-resolved fluorescence; lifetime-based pH sensing; microplate assay; glycolytic flux; mitochondrial dysfunction; screening

Materials & Methods:
PC12 cells were differentiated for 3 to 5 days in RPMI supplemented with 1% HS 100 IU/ml penicillin 100 lg/ml streptomycin and 100 ng/ml nerve growth factor on collagen/poly-D-lysinecoated glass-bottom mini-dishes (MatTek Ashland MA USA). One full day (24 h) before measurement cells were transfected with the memCase12 plasmid (Evrogen JSC Russia) a mitochondria- targeted version of Case12 [25] using Lipofectamine and Opti- MEM I (Invitrogen Carlsbad CA USA) as per the manufacturer’s procedure. Cells were imaged on an Olympus FV1000 confocal laser scanning microscope with excitation at 488 nm (1.5% of laser power) and emission collected at 500 to 550 nm. Fluorescence images were collected with a 60 oil immersion objective in two planes using 0.5 lm step and 20-s intervals. The resulting zstacked images were analyzed using FV1000 Viewer software (Olympus) and Adobe Photoshop and Illustrator.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
PC12 cells