TY - JOUR
T1 - Imaging of cellular oxygen and analysis of metabolic responses of mammalian cells.
AU - Fercher, Andreas
AU - O'Riordan, Tomas C.
AU - Zhdanov, Alexander V.
AU - Dmitriev, Ruslan I.
AU - Papkovsky, Dmitri B.
PY - 2010
Y1 - 2010
N2 - Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca(2+), H(+)), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite - cellular oxygen - is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O(2) in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O(2) concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given.
AB - Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca(2+), H(+)), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite - cellular oxygen - is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O(2) in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O(2) concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given.
UR - https://www.scopus.com/pages/publications/77449125086
U2 - 10.1007/978-1-60761-404-3_16
DO - 10.1007/978-1-60761-404-3_16
M3 - Article
C2 - 19957136
AN - SCOPUS:77449125086
SN - 1064-3745
VL - 591
SP - 257
EP - 273
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -