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).