Authors
Ivan Zanoni, Renato Ostuni, and Francesca Granucci
Summary
Protocol for calcium transients measurement in dendritic cells used in our Nature paper. Intracellular calcium concentration was determined by a fluorimetric ratio technique, an approach that overcomes possible problems of uncertainty related to the calibration or uneven distribution of fluorescent calcium indicators. A direct optical microscope (Olympus, BX51) with a two-photon Ti-Sapph laser source (720 nm wavelength; Mai Tai, SpectraPhysics) was used for indo-1 excitation. The fluorescence signals emitted by indo-1-loaded cells were digitized at 200 Hz and recorded every 0.5-0.8 s. The ratio of fluorescence emissions at 400 nm/bp to those at 500 nm/bp was recorded (R400/500) and used as an index of intracellular calcium concentration. Data were then normalized to baseline. In some cases, cells were analyzed in calcium-free PBS or calcium-free PBS supplemented with 50 nM thapsigargin (Sigma Aldrich).Further details
The protocol was published on Protocol Exchange on 18 June 2009. To see the entire protocol, click on the source link.Advertisement
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